Dopamine receptors and genes

ABSTRACT

A mammalian D 2  dopamine receptor gene has been cloned. Thus, DNA sequences encoding all or a part of the dopamine receptor are provided, as well as the corresponding polypeptide sequences and methods for producing the same both synthetically and via expression of a corresponding sequence from a host transformed with a suitable vector carrying the corresponding DNA sequence. The various structural information provided by this invention enables the preparation of labeled or unlabeled immunospecific species, particularly antibodies, as well as nucleic acid probes labeled in conventional fashion. Pharmaceutical compositions and methods of using various products of this invention are also provided.

[0001] This application is a continuation of U.S. Ser. No. 07/438,544,filed Nov. 20, 1989, now abandoned, which was a continuation-in-part ofU.S. Ser. No. 07/273,373, filed Nov. 18, 1988, now abandoned.

BACKGROUND OF THE INVENTION

[0002] This invention relates to dopamine receptors from mammalianspecies and the corresponding genes. In particular, it relates to theisolation, sequencing and/or cloning of D₂ dopamine receptor genes, thesynthesis of D₂ dopamine receptors by transformed cells, and themanufacture and use of a variety of novel products enabled by theidentification and cloning of DNA encoding dopamine receptors.

[0003] Dopamine receptors in general have been implicated in a largenumber of neurological and other disorders, including, for example,movement disorders, schizophrenia, drug addiction, Parkinson's disease,Tourette syndrome, Tardive Dyskinesia, and many others. As a result, thedopamine receptor has been the subject of numerous pharmacological andbiochemical studies.

[0004] In general, dopamine receptors can be classified into D₁ and D₂subtypes based on pharmacological and biochemical characteristics (1,2). The D₂ dopamine receptor has been implicated in the pathophysiologyand treatment of the mentioned disorders. In addition, it is known thatthe D₂ dopamine receptor interacts with guanine nucleotide bindingproteins to modulate second messenger systems (6, 7).

[0005] Despite the heavy emphasis placed on elucidation of the existenceand properties of the dopamine receptor and its gene, identification,isolation and cloning have been inaccessible heretofore. This is truedespite the knowledge that other members of the family of receptors thatare coupled to G proteins share a significant similarity in primaryamino acid sequence and exhibit an archetypical topology predicted toconsist of seven putative transmembrane domains (8). Regarding theserotonin receptor, e.g., see Julius et al., Science, Vol. 241, 558(1988).

SUMMARY OF THE INVENTION

[0006] This invention has successfully identified, isolated and clonedmammalian, including human, D₂ dopamine receptor gene sequences andproduced the encoded dopamine receptor. The corresponding polypeptidehas been synthesized. Sequences of both the gene and the polypeptidehave been determined. The invention also provides a variety of new anduseful nucleic acid, cell line, vector, and polypeptide and medicinalproducts, inter alia, as well as methods of using these.

[0007] Thus, this invention relates to an isolated DNA sequence, anidentified portion of which is a structural gene which encodes apolypeptide having the biological activity of a mammalian D₂ dopaminereceptor. In particular, it relates to an isolated DNA sequence whichwill hybridize to a DNA sequence encoding a mammalian D₂ dopaminereceptor. It also relates to fragments, variants and mutants of suchsequences, particularly those which also encode a polypeptide havingbiological activity of a mammalian dopamine receptor, most particularlya mammalian D₂ dopamine receptor. In a preferred aspect, the dopaminereceptor is human. In another preferred aspect, the sequence is that ofrat D₂ dopamine receptor as shown in FIG. 1. Of course, the nucleicacids of this invention also include complementary strands of theforegoing, as well as sequences differing therefrom by codon degeneracyand sequences which hybridize with the aforementioned sequences. Inother preferred aspects, this invention includes nucleic acid sequencesand fragments useful as oligonucleotide probes, preferably labelled witha detectable moiety such as a radioactive or biotin label. For example,such probes can hybridize with DNA encoding a polypeptide having thebiological activity of a D₂ dopamine receptor or with DNA associatedtherewith, e.g., DNA providing control of a D₂ dopamine receptor gene orintrons thereof, etc. DNA of this invention can also be part of avector.

[0008] The invention also involves cells transformed with vectors ofthis invention as well as methods of culturing these cells tomanufacture polypeptides, e.g., having the biological activity of a D₂dopamine receptor. Preferably, the cells are of mammalian origin whenused in such methods.

[0009] The invention also relates to polypeptides encoded by theforegoing nucleic acid sequences, especially to isolated mammaliandopamine receptors, preferably of human origin. The invention furtherrelates to polypeptides which are mutants or variants of such receptors,preferably those wherein one or more amino acids are substituted for,inserted into and/or deleted from the receptor, especially those mutantswhich retain the biological activity of a dopamine receptor. Thisinvention also relates to antibodies, preferably labelled, and mostpreferably monoclonal, capable of binding a dopamine receptor amino acidsequence, preferably wherein the latter is human, or a fragment of suchan antibody.

[0010] The invention further relates to compositions comprising one ofthe various products mentioned above and, typically, a pharmaceuticallyacceptable carrier as well as to methods of employing these products toachieve a wide variety of utilitarian results.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Various other objects, features and attendant advantages of thepresent invention will be more fully appreciated as the same becomesbetter understood when considered in conjunction with the accompanyingdrawings, and wherein:

[0012]FIG. 1 shows the nucleotide sequence for the RGB-2 cDNA anddeduced amino acid sequence of the rat D₂ dopamine receptor. Thenucleotide sequence is numbered beginning with the first methionine ofthe long open reading frame. Nucleotide numbering appears beneath thenucleotide sequence at the right-hand end of each line. The deducedamino acid sequence is shown above the nucleotide sequence. The doubleunderline denotes the small open reading frame in the 5′ untranslatedregion. Postulated N-linked glycosylation sites are indicated byasterisks. Putative protein kinase A phosphorylation sites have a lineabove them. The intron splice junction is designated by an arrow. Thepoly A adenylation site is underlined.

[0013]FIG. 2 shows the alignment of the amino acid sequences of the ratD₂ dopamine receptor, the hamster β₂-adrenergic receptor, the humanα₂-adrenergic receptor, the human G-21 (serotonin 1a) receptor, theporcine muscarinic M receptor and the bovine substance K receptor. Aminoacids enclosed in solid lines and shaded represent residues that areidentical in at least two other sequences when compared to the D₂dopamine receptor. The putative transmembrane domains are indicated bybrackets labeled by Roman numerals. The numbers of residues in thevariable third cytoplasmic loop and at the C-terminus are inparentheses.

[0014]FIG. 3 shows a Northern blot analysis of RGB-2 transcripts in ratbrain and pituitary. Each lane contained 20 μg of total RNA. Numbers onthe right indicate molecular weight as determined from RNA size markers(BRL). A: olfactory bulb; B: hippocampus; C: cerebellum; D: posteriorcortex; E: anterior cortex; F: thalamus; G: hypothalamus; H: medulla; I:amygdala; J: mesencephalon; K: septum; L: posterior basal ganglia; M:anterior basal ganglia; N: neurointermediate lobe of pituitary; O:anterior lobe of the pituitary.

[0015]FIG. 4 illustrates the binding of ³H-spiperone to membranes fromL-RGB2Zem-1 cells.

[0016] a) 1: Saturation isotherms of the specific binding of³H-spiperone to membranes prepared from L-RGB2Zem-1 cells and ratstriatum. Results are shown from one of four independent experiments.

[0017] 2: Scatchard transformation of the data.

[0018] b) Competitions curves using L-RGB2Zem-1 membranes.Representative curves are shown for inhibition of specifically bound³H-spiperone by drugs in membranes from L-RGB2Zem-1 cells. Each drug wastested 3 times.

[0019] c) Table of K_(i) values for L-RGB2Zem-1 and rat striatum.Results are geometric means of 3 experiments in which 0.5 nM³H-spiperone was inhibited by various concentrations of unlabeled drug.For some drugs, inhibition curves in rat striatal tissue were fit bestby assuming the presence of two classes of binding sites. Theproportions of binding sites with high or low affinity for inhibitor areshown in parentheses. K_(i) values for the class of binding sitesrepresenting 10-25% of specific binding were calculated by assuming thatthe radioligand was binding to serotonin receptors with a Kd value of 1nM.

[0020]FIG. 5 shows (a) a hydrophobicity plot of the amino acid sequenceshown in FIG. 1; and (b) a hydophobicity plot of the amino acid sequenceof the β₂-adrenergic receptor. The transmembrane regions are marked bythe Roman numerals.

[0021]FIG. 6 shows a calculated restriction map of a 2477 base EcoRIfragment of the nucleic acid sequence shown in FIG. 1.

[0022]FIG. 7 shows a partial sequence of a human D₂ dopamine receptor,the middle amino acid sequence shown being the correct one.

[0023]FIG. 8 shows a saturation analysis of specific [³H]spiroperidolbinding to LZR1 and LZR2 cells. Results shown are from representativeexperiments in which LZR1 and LZR2 cells were grown in control growthmedium or medium to which zinc sulfate had been added, as indicated.Data are plotted as specifically bound radioligand divided by thecorrected free concentration of radioligand (total added minus totalbound), versus specifically bound radioligand. For zinc treatments, 100μM zinc sulfate was added to growth medium for 16 hours. Both controland zinc-treated cells were then washed and grown in control medium forone day before harvesting. In the experiments shown, B_(max) valuesdetermined by nonlinear regressions analysis were 876 and 914 fmol/mg ofprotein for control LZR1 cells or LZR1 cells treated with zinc,respectively. B_(max) values for control or zinc-treated LZR2 cells were385 and 593 fmol/gm of protein, respectively.

[0024]FIG. 9 shows the inhibition of radioligand binding by agonists.Results are plotted as specific binding, expressed as a percentage ofspecific binding in the absence of competing drug, versus the log of theconcentration of competiting drug. Membranes were prepared from LZR1cells as described in the text.

[0025] A. Curves from a single experiment are shown for inhibition ofthe binding of [³H]spiroperidol by agonists. Each drug was tested twice.In this experiment, the free concentration of [³H]spiroperidol was 230pM, and the K₀ value for [³H]spiroperidol was 60 pM. K₁ values and Hillcoefficients in this experiment were 5 nM and 1.05 for bromocriptine,respectively, 790 nM and 0.89 for (−)3-PPP, 8 μM and 1.0 for quinpirole,31 μM and 1.05 for (+)3-PPP, and 0.3 mM and 0.72 for LY181990.

[0026] B. Results are shown from one of four independent experiments inwhich the effect of GTP and NaCl on the inhibition of [³H]spiroperidolbinding by DA was determined. Concentrations of [³H]spiroperidol rangedfrom 323 to 498 pM. In this experiment, the concentration of radioligandwas 323 pM. Open circles represent inhibition by DA in the presence of0.1 mM GTP and 120 mM NaCl, whereas closed circles represent inhibitionin the absence of added GTP and NaCl. IC₅₀ values and Hill coefficientsin this experiment were 29 μM and 0.65, respectively, in the absence and115 μM and 1.03 in the presence of GTP and NaCl.

[0027]FIG. 10 shows the inhibition of adenylate cyclase activity in LZR1cells. Agonists were tested for inhibition of adenylate cyclase activityin membranes prepared from LZR1 cells. Approximately 50 to 100 μg ofprotein was used in each assay. Results are shown as [³P]cAMP/mg ofprotein/min., expressed as a percentage of total activity in thepresence of 10 μM forskolin. Representative dose-response curves areshown for six drugs, each tested at least three times. Data are plottedas enzyme activity versus the log of the concentration of drug. No curveis plotted for the data for (−)3-PPP, since no inhibition was observed.In the experiments shown in this figure, basal and forskolin-stimulatedactivity ranged from approximately 0.8 to 1.5 and 8.5 to 15.8 pmol/mg ofprotein/min., respectively.

[0028]FIG. 11 shows the blockade of DA-sensitive adenylate cyclase.Results shown are means of three experiments ±SE, plotted as thepercentage of total activity in the presence of 10 μM forskolin.Forskolin (FSK) was present in all the experiments shown, together with10 μM dopamine (DA) or DA and 10 μM (+)-butaclamol (BUT) as indicated.Some cells were treated with pertussins toxin (PT) before harvesting fordetermination of enzyme activity. Basal activity in control andPT-treated cells was 1.2±0.07 and 1.6±0.15 pmol/mg of protein/min.,respectively. Total forskolin-stimulated activity in control cells (FSK)was 11.9±1.0 pmol/mg of protein/min. *p<0.05 compared to FSK in controlcells, as determined by a t test for paired means.

[0029]FIG. 12 shows the reversal of dopamine inhibition by pertussistoxin pretreatment. Data presented for membrane adenylate cyclaseactivity represent means (x) with standard error (S.E.) and % inhibition(% Inh.) below. % Inhibition was calculated from the equation100×[1−(S−B/I−B₁)] where B, S, and I are values of basal activity,activity in the presence of stimulator (S) or inhibitor (I),respectively and B₁ is basal activity in the presence of inhibitor.Results were obtained from parallel assays in controls and cellspretreated (16 h, 50 ng/ml) with pertussis toxin (indicated as +P.T.).

[0030] (A) Adenylate Cyclase. Membranes for cyclase assay were exposedacutely to 10 μM forskolin (FSK) or 100 μM dopamine (DA), and adenylatecyclase activity expressed as pmol/mg protein/min.

[0031] (B) Intracellular cAMP. Cells were treated acutely with VIP (200nM) and dopamine (1 μM) and cAMP accumulation (expressed as pmol/dish)was measured in cell extracts.

[0032] (C) Extracellular cAMP. Media samples from the same dishes ofcells were assayed for cAMP accumulation expressed as pmol/dish.

[0033]FIG. 13 shows the expression of specific dopamine-D₂ receptor mRNAand specific binding in GH₄ZR₇ transfectant cells.

[0034] (A) Northern blot analysis of GH₄C₁ cell total RNA′ (20 μg/lane).Y-axis indicate the migration of RNA molecular weight standards (kb).

[0035] (B) (1) Specific binding of ³H-spiperone to membranes preparedfrom GH₄ZR₇ cells was characterized by saturation analysis (see“Experimental Procedures”, Example 2). Data from one of four independentexperiments are plotted as specifically bound radioligand (ordinate)versus corrected free radioligand concentration (total added minus totalbound). Calculated K₀ and B_(max) values for this experiment were 60 pMand 1165 fmol/mg protein.

[0036] (B)(2) Transformation of the data by the method of Scatchardwhich are plotted as specific bound/free (Y-axis) vs. specific boundconcentrations of ³H-spiperone (X-axis).

[0037] (C) Displacement of specific ³H-spiperone binding by dopamine:effect of GTP/NaCl. GH₄ZR₇ cell membranes were incubated with³H-spiperone (0.47 nM) and indicated concentrations of dopamine (X-axis)in the absence (o) or presence (•) of 100 μM GTP/120 mM NaCl. Resultsare shown for one of four experiments. Calculated IC₅₀ and Hillcoefficient values for dopamine in the experiment shown were 16 μM and0.61 in the absence of GTP/NaCl, and 56 μM and 0.85 in the presence ofGTP/NaCl.

[0038]FIG. 14 shows the inhibition of cAMP accumulation and PRL releaseby dopamine in GH₄ZR₇ cells. Incubations were performed in triplicate asdescribed in “Experimental Procedures”, Example 2.

[0039] (A) Inhibition of extracellular cAMP accumulation by dopamine.Parallel dishes of GH₄C₁ and GH₄ZR₇ cells were incubated withconcentrations of VIP, dopamine (D), and (−)-sulpiride (−S) of 250 nM,10 μM and 5 μM, respectively. Untreated controls are denoted as “C”.Media were collected and assayed for cAMP (ordinate) expressed aspmol/dish.

[0040] (B) Inhibition of intracellular cAMP accumulation by dopamine inGH₄ZR₇ cells. Cell extracts were assayed for cAMP, expressed on theordinate. Drug concentrations were as in (A), except (+)-sulpiride (+S),5 μM.

[0041] (C) Inhibition of stimulated PRL release by dopamine in GH₄ZR₇cells. Media samples were assayed for PRL (ordinate) after the indicatedtreatments. The concentrations of VIP, TRH, dopamine (D), and(−)-sulpiride (−S) were 200 nM, 200 nM, 100 nM, and 2 μM, respectively.

[0042]FIG. 15 shows dose-response relations for dopamine inhibition ofbasal and VIP-enhanced cAMP accumulation in GH₄ZR₇ cells.

[0043] (A) Basal intra-(•) and extracellular (o) cAMP accumulation inthe presence of indicated concentrations of dopamine. Basal cAMP levelsin the absence of dopamine were 22±6 pmol/dish (intracellular) and12.4±0.6 pmol/dish (extracellular). EC₅₀ values for dopamine actionswere 4.9 nM (intracellular) and 8.5 nM (extracellular).

[0044] (B) VIP-enhanced intra-(•) and extracellular (o) cAMPaccumulation in the presence of indicated dopamine concentrations. VIP(250 nM)-enhanced levels of intra- and extracellular cAMP (in theabsence of dopamine) were 145±1.2 pmol/dish and 146±2.8 pmol/dish, andbasal cAMP levels were 35±1.6 pmol/dish and 15±0.2 pmol/dish,respectively. EC₅₀ values for dopamine inhibition were 5.5 nM(intracellular) and 5.8 nM (extracellular).

[0045]FIG. 16 shows the specific blockade of dopamine-induced inhibitionof VIP-enhanced cAMP accumulation. GH₄ZR₇ cells were incubated in FATmedium (see “Methods”, Example 2) in the presence of 250 nM VIP and 100nM dopamine, and indicated concentrations of various dopamineantagonists, and extracellular levels of cAMP measured. Data are plottedas percent of maximal cAMP levels versus the logarithm of indicatedconcentrations of dopamine antagonists. The standard error of triplicatedeterminations was less than 8%. Basal and VIP-enhanced cAMP levels (inthe absence of dopamine and antagonists) were 25.8±1.6 pmol/dish and214±14 pmol/dish. IC₅₀ and estimated K_(i) values for antagonism ofdopamine actions were: spiperone, 0.56 nM and 30 pM; (+)-butaclamol, 4.5nM and 0.2 nM; (−)-sulpiride 38 nM and 1.8 nM; SCH23390, >1 μM;(−)-butaclamol, >10 μM. Estimated K_(I) were calculated from theequation K_(I)−IC₅₀/(1+([DA]/EC₅₀)), where [DA] is 100 nM and the EC₅₀for dopamine is 6 nM. Spiperone, (+)-butaclamol and (−)-sulpiride alonedid not alter basal cAMP levels.

[0046]FIG. 17 shows the inhibition of adenylate cyclase by dopamine-D₂agonists. Inhibition of adenylate cyclase activity was assessed in thepresence of 10 μM forskolin. Data are plotted as the mean of triplicateassays, with enzyme activity expressed as a percentage of total activityversus the logarithm of drug concentration. Average basal adenylatecyclase activity was 4.6±0.2 pmol/mg protein/min and totalforskolin-stimulated activity was 63.8±0.2 pmol/mg protein/min. EC₅₀values and maximal inhibition for the experiments shown were 79 nM and57%, respectively, for dopamine, 200 nM and 49% for quinpirole, 5 nM and23% for bromocryptine, and 600 μM and 40% for (+)-3-PPP.

[0047]FIG. 18 shows the nucleotide sequence of the human pituitarydopamine D₂ receptor cDNA. The deduced amino acid sequence is indicatedabove the human cDNA. Below is the nucleotide sequence of the cloned ratcDNA (see Reference 9, Example 3) and the amino acids which differbetween the two clones. Boxed regions, numbered I-VII, represent theputative transmembrane domains. Triangles indicate the exon/intronsplice junctions; a period is one missing base pair; asterisks identifypotential N-linked glycosylation sites and targets of protein kinase Aphosphorylation are underlined. The polyadenylation signal is doubleunderlined.

[0048]FIG. 19 shows competition curves of [3H]-domperidone binding toL-hPitD₂Zem membranes. The radioligand was used at a concentration of 1nM, and specific binding was defined using 1 μM (+) butaclamol. Data areshown for one of three experiments.

[0049]FIG. 20 shows Southern blot of human genomic DNA. The genomicBamHI 1.6-kb fragment containing exon 7 was prepared from λHD2G1 andused as probe (specific activity, 2×10⁸ cpm). DNA was digested withBamHI (lane 1), Bg1II (lane 2), BamHI/Bg1II (lane 3), and HindIII (lane4).

[0050]FIG. 21 is a schematic representation of the human dopamine D₂receptor gene and pituitary cDNA.

[0051] (a) Restriction map of the two overlapping genomic phase, λhD2G1and λhD2G2; A, ApaLI; B, BamHI; Bg, Bg1II; H, HindIII.

[0052] (b) Diagram of the human gene locus DRD2. Exons, indicated by theboxes, are numbered. The solid boxes indicate regions of coding sequenceand open boxes, non-translated sequence. The genomic sequencing strategyis expanded above the gene. These regions are not drawn to scale. DNAsequences (+ indicates sense and −, antisense sequence) were read fromthe bottom to the top of each ladder in the directions indicated by thearrows (pointing to the right, 5′ to 3′ and to the left, 3′ to 5′). The87-bp exon is No. 5. Intron sizes were determined by Southern blottingand restriction mapping and are accurate to within 10 percent, with oneexception: intron 1 is accurate to within 20 percent.

[0053] (c) The structure of the human pituitary cDNA. Exon/intron splicejunctions are indicated by triangles. Regions encoding transmembranedomains I-VII are enclosed by boxes containing wavy lines. The 87-bpsequence is striped. The sites for translation initiation (ATG) andtermination (TAG) are indicated, as is the polyadenylation signalsequence (AATAAAA).

[0054]FIG. 22 shows a comparison of IC₅₀ values (nM) for L-hPitD2Zem,L-RGB2Zem1 and rat striatum.

[0055]FIG. 23 shows the exon/intron junctions in the human dopamine D₂receptor gene.

DISCUSSION

[0056] This invention takes advantage in a unique way of nucleotidesequence similarities among members of a gene family coding forreceptors that are coupled to G proteins. By using a unique hamsterβ₂-adrenergic receptor (β₂AR) gene as a hybridization probe, a cDNAencoding the rat D₂ dopamine receptor was identified and isolated. Thereceptor has been characterized on the basis of three criteria: 1) thededuced amino acid sequence which reveals that it is a member of thefamily of G protein-coupled receptors, 2) the tissue distribution of thecorresponding mRNA which parallels that known for the D₂ dopaminereceptor, and 3) the pharmacological profile of Ltk-cells transformedwith the cDNA.

[0057] A rat genomic library was screened under low-stringencyhybridization conditions with a nick-translated 1.3 kb HindIII fragmentcontaining most of the coding region of the hamster β₂AR gene. Thehamster β₂AR receptor gene was cloned from a partial hamster lung λgt10genomic DNA library (constructed from size fractionated (5-7 kb) EcoRIdigested DNA) with two oligonucleotide probes (30-mer,TCTGCTTTCAATCCCCTCATCTACTGTCGG; 40-mer,CTATCTTCTGGAGCTGCCTTTTGGCCACCTGGAAGACCCT) designed from the sequence ofDixon et al. (9). The 1.3 kb HindIII fragment of the hamster β₂AR genewhich contains most of the coding sequence of that gene was labeled bynick translation and used to probe a rat genomic DNA library in thecommercially available phage EMBL3. The library was transferred toColony Plaque Screen filters (NEN) and screened with the ³²P labeledprobe using the following hybridization conditions: 25% formamide,5×SSC, 5× Deńhardts, 0.1% sodium pyrophosphate, 1% SDS and salmon spermDNA (100 μg/ml) at 37° C. Filters were washed in 2× $SC and 0.1% SDS at55° C.

[0058] Several clones were found to hybridize to the hamster probe usingthese conditions. One clone, called RGB-2, was found to have a 0.8 kbEcoRI-PstI fragment that hybridized to the hamster β₂AR probe inSouthern blot analysis. This fragment was sequenced and shown to have astretch of nucleotides with a high degree of identity (32 out of 40bases) to the nucleotide sequence of transmembrane domain VII of thehamster β₂AR. One of the possible reading frames demonstrated asignificant similarity to the amino acid sequence of transmembranedomains VI and VII of the hamster β₂AR. Within this genomic fragmentthere is also a 3′intron splice site (10) and 400 bp of putativeintronic sequence.

[0059] The 0.8 kb EcoRI-PstI fragment (nick translated) was used toprobe a rat brain cDNA library in λgt10 with the same hybridizationconditions as above except that 50% formamide was used. Washing of thefilters was performed in 0.2×SSC and 0.1% SDS at 65° C. Under these highstringency hybridization conditions, two positive clones of about 2.5 kbin size were identified from a library of 500,000 clones. DNA sequencewas obtained from both strands by the Sanger dideoxy chain determinationmethod using Sequenase (U.S. Biochemical) (26). Sequence and restrictionanalysis indicated that the two clones were replicas of a single cloneand contained the sequence of the genomic fragment that had been used asa probe. When the RGB-2 cDNA was used as a hybridization probe inNorthern blot analysis of mRNA isolated from rat brain, a band ofapproximately 2.5 kb was detected. This finding indicated that the RGB-2clone was nearly full length.

[0060]FIG. 1 shows the nucleotide sequence of 2455 bases for the RGB-2cDNA. The longest open reading frame in this cDNA codes for a 415 aminoacid protein (relative molecular weight (Mr=47,064)) also shown in thefigure. This molecular weight is similar to that reported for thedeglycosylated form of the D₂ dopamine receptor as determined by SDSpolyacrylamide gel electrophoresis (11). An in-frame dipeptide which is36 bases upstream from the putative initiation site is found in the 5′untranslated region of the RGB-2 cDNA. A small open reading frame hasbeen observed in the 5′ untranslated sequence of the β₂AR mRNA (9).

[0061] Several structural features of the protein deduced from the RGB-2cDNA demonstrate that it belongs to the family of G protein-coupledreceptors. The hydrophobicity plot of the protein sequence (FIG. 5)shows the existence of seven stretches of hydrophobic amino acids whichcould represent seven transmembrane domains (8). Moreover, the primaryamino acid sequence of RGB-2 shows a high degree of similarity withother G protein-coupled receptors (FIG. 2). The regions of greatestamino acid identity are clustered within the putative transmembranedomains. Within these domains the RGB-2 protein has a sequence identityof 50% with the human α₂-adrenergic receptor (12), 42% with the humanG-21 receptor (13), 38% with the hamster β₂AR-adrenergic receptor (9),27% with the porcine M₁ receptor (14), and 25% with the bovine substanceK-receptor (15).

[0062] Thirdly, RGB-2 has several structural characteristics common tothe members of the family of G protein-coupled receptors. There arethree consensus sequences for N-linked glycosylation in the N-terminuswith no signal sequence. The Asp 80 found in transmembrane domain II isconserved in all known G protein-coupled receptors. In transmembranedomain III there is Asp 114 for which a corresponding Asp residue isfound only in receptors that bind cationic amines (16). Phosphorylationhas been proposed as a means of regulating receptor function (17). Apotential site for phosphorylation by protein kinase A exists at Ser 228in the third cytoplasmic loop. In the C-terminal portion of therhodopsins and β-adrenergic receptors are found many Ser and Thrresidues which are potential substrates for receptor kinases (18). Incontrast, the short C-terminus of RGB-2 has no Ser and Thr residues.However, as is the case for the α₂-adrenergic receptor, RGB-2 has manySer and Thr residues (22 residues) in the third cytoplasmic loop whichcould serve as phosphorylation substrates.

[0063] RGB-2 contains a large cytoplasmic loop (135 amino acids) betweentransmembrane domains V and VI with a short C-terminus (14 amino acids).This structural organization is similar to other receptors which arecoupled by G_(i) (inhibitory G protein) such as the ₂-adrenergicreceptor and the M₂ muscarinic receptor. Unlike the members of theadrenergic and muscarinic receptor families, the RGB-2 gene has at leastone intron in its coding sequence which is located in transmembranedomain VI.

[0064] As a first step towards determining the identity of RGB-2, thetissue distribution of the RGB-2 mRNA was examined by Northern blotanalysis (FIG. 3).

[0065] Brain tissue was dissected from male Sprague Dawley rats and RNAisolated according to Chirgwin et al. (27) and Ullrich et al. (28). ForNorthern blotting, RNA was denatured using glyoxal and DMSO and run on1.2% agarose gels. After electrophoresis, RNA was blotted onto a nylonmembrane (N-Bond, Amersham) and baked for 2 hours at 80° C. Themembranes were prehybridized in 50% formamide, 0.2% PVP (M.W. 40,000),0.2% ficoll (M.W. 400,000), 0.05 M Tris pH 7.5, 1M NaCl, 0.1% PPi, 1%SDS and denatured salmon sperm DNA (100 μg/ml) for 16 hrs at 42° C. Arandom primed ³²P-labeled fragment (1-2 10⁸ dpm/μg) from the 1.6 kbBamHI-BglII fragment of RGB-2 which contains the coding region of thisclone was used at 10⁷ dpm/ml in the hybridization solution from above toprobe the filters overnight at 42° C. The blots were washed twice in2×SSC and 0.1% SDS at 65° C. for 10 min., twice in 0.5×SSC and 0.1% SDSat RT for 15 min. and once in 10.1×SSC and 0.1% SDS at 65° C. for 15min. Blots were exposed overnight at −70° C. to X-ray film with anintensifying screen.

[0066] The RGB-2 mRNA is expressed at different levels in variousregions of the rat brain with the basal ganglia showing the highestconcentration. Furthermore, the RGB-2 mRNA was found in high amounts inthe neurointermediate lobe of the pituitary gland of the rat and to alesser degree in the anterior lobe of this gland. The expression patternof the RGB-2 mRNA is strikingly similar to the distribution of the D₂dopamine receptor as determined by receptor autoradiography and bindingstudies of tissue preparations (19).

[0067] In order to study the pharmacological characteristics of thereceptor encoded by RGB-2, the cDNA was expressed in eucaryotic cells.The full RGB-2 cDNA was cloned into the eucaryotic expression vectorpZem3 (20) which initiates transcription from the mouse metallothioneinpromoter (21). This plasmid was cotransfected with the selectableneomycin phosphotransferase gene (pRSVneo) into the Ltk-mouse fibroblastcell line by the standard CaP₄ precipitation technique (21). Cells wereselected in 350 μg/ml of G418. Transfectants were isolated and checkedfor expression of RGB-2 mRNA by Northern blot analysis. A cell lineexpressing RGB-2 designated L-RGB2Zem-1 was isolated. The RGB-2 mRNA wasnot detectable in the parent Ltk-cell line.

[0068] Since the RGB-2 mRNA displayed the tissue distribution expectedof the D₂ dopamine receptor, a pharmacological study was performed ofthe L-RGB2Zem-1 cell line, native Ltk-cells and rat striatum using theD₂ ligand ³H-spiperone.

[0069] Membranes were prepared by homogenizing cells with a Douncehomogenizer at 4° C. in 0.25 M sucrose, 25 mM Tris pH 7.4, 6 mMMgCl_(2, 1) mM EDTA. The homogenizing solution was centrifuged at 800×gfor 10 min. and the pellet was subjected to a second homogenization andcentrifugation as before. The supernatants were pooled and centrifugedat 200,000×g for 1 hour. The pellet of this centrifugation wasresuspended in 25 mM Tris pH 7.4, 6 mM MgCl₂, 1 mM EDTA at approximately250 μg protein/ml and stored in small aliquots at −70° C. Radioligandbinding assays were carried out in duplicate in a volume of 2 ml(saturation analyses) or 1 ml (inhibition curves) containing (finalconcentration): 50 mM Tris, pH 7.4, 0.9% NaCl, 0.025% ascorbic acid,0.001% bovine serum albumin, ³H-spiperone (Amersham, 95 Ci/mmol) andappropriate drugs. In some experiments 100 uM guanosine 5′-triphosphatewas included. (+)-Butaclamol (2 uM) was used to define nonspecificbinding. Incubations were initiated by the addition of 15-40 μg ofprotein, carried out at 37° C. for 50 minutes, and stopped by theaddition of 10 ml of ice-cold wash buffer (10 mM Tris, pH=7.4, and 0.9%NaCl) to each assay. The samples were filtered through glass-fiberfilters (Schleicher and Schuell No. 30) and washed with an additional 10ml of wash buffer.

[0070] The radioactivity retained on the filter was counted using aBeckman LS 1701 scintillation counter. Data were analyzed as previouslydescribed (29) except that curves were drawn using the data analysisprogram Enzfitter. The resulting IC₅₀ values were converted to K_(i)values by the method of Cheng and Prusoff (30).

[0071] Membranes prepared from control Ltk-cells showed no(+)-butaclamol- or sulpiride-displaceable binding of ³H-spiperone.Binding of ³H-spiperone to membranes prepared from L-RGB2Zem-1 cells wassaturable with a Kd value of 48 pM (FIG. 4a). This value agrees withthat observed for binding of ³H-spiperone to rat striatal membranes inparallel experiments (52 pM). In the experiment shown in FIG. 4a, Kd andBmax values for membranes prepared from L-RGB2Zem-1 were 40 pm and 876fmol/mg of protein, whereas the corresponding values in striatalmembranes were 37 pm and 547 fmol/mg of protein. The density of bindingsites as determined in four experiments was 945 fmol/mg of protein inL-RGB2Zem-1 membranes and 454 fmol/mg of protein in rat striatalmembranes.

[0072] The binding of ³H-spiperone to membranes from L-RGB2Zem-1 cellswas inhibited by a number of drugs and the resulting K_(i) valuesclosely matched those obtained using striatal membranes (FIGS. 4b, 4 c).The D₂ antagonists (+)-butaclamol and haloperidol were the most potentinhibitors, followed by sulpiride. The D₁ dopamine antagonist SCH 23390and the serotonin 5HT₂ antagonist ketanserin were much less potent atblocking ³H-spiperone binding. The binding appeared to bestereoselective as (+)-butaclamol was much more potent than(−)-butaclamol at inhibiting binding. In these experiments the absoluteaffinities of dopaminergic antagonists and the rank order of potency ofthe drugs(spiperone>(+)-butaclamol>haloperidol>sulpiride>>(−)-butaclamol) agreeclosely with previously published values for the D₂ dopamine receptor(2).

[0073] All binding data for L-RGB2Zem-1 membranes were fit best byassuming the presence of only one class of binding sites. On the otherhand, inhibition by several drugs of ³H-spiperone binding to ratstriatal membranes was fit best by assuming the presence of two classesof binding sites. Thus, SCH 23390 and ketanserin inhibited 10-20% of³H-spiperone binding to rat striatal membranes with high affinity (FIG.4c). In rat striatal membranes, inhibition of radioligand binding bysulpiride was fit best by one class of binding sites, but 10-15% of the(+)-butaclamol-displaceable binding was not inhibited by sulpiride atthe concentrations used. It seems likely that the binding sites withhigh affinity for ketanserin and SCH 23390 and which are not displacedby sulpiride represent binding of ³H-spiperone to 5HT₂ serotoninreceptors in rat striatal membranes. Binding of SCH 23390 to 5HT₂receptors has been described previously (22). In rat striatal membranes,the apparent affinity of drugs for D₂ dopamine receptors, the class ofbinding sites comprising 80-90% of ³H-spiperone binding, wasindistinguishable from the apparent affinity of drugs to membranesprepared from L-RGB2Zem-1 cells (FIG. 4c).

[0074] The physiological effects of stimulation of D₂ dopamine receptorsappear to be mediated by G_(i) (6). Inhibition of agonist binding to D₂dopamine receptors by GTP is thought to be due to GTP-induceddissociation of a receptor-G_(i) complex which has a high affinity foragonist (23, 24). Although ³H-spiperone binding was inhibited by theagonist dopamine with a K_(i) of 17 um in the L-RGB2Zem-1 membranes,this dopamine binding was not responsive to the addition of GTP.However, this finding is consistent with the reported lack of G_(i) in Lcells (25). The pharmacological data presented here proves that thebinding profile of the D₂ dopamine receptor is found in Ltk-cellsexpressing the RGB-2 cDNA.

[0075] The foregoing data show that, when transfected into eucaryoticcells, the RGB-2 cDNA directs the expression of a D₂ dopamine bindingprotein. Since the mRNA corresponding to this cDNA is localized intissues where the D₂ dopamine receptor is known to be present and sincethis mRNA codes for a protein which has all the expected characteristicsof a G protein-coupled receptor, inter alia, RGB-2 is a clone for therat D₂ dopamine receptor.

[0076] The nucleic acid sequence shown in FIG. 1 can be inserted into awide variety of conventional and preferably commercially availableplasmids, e.g., using EcoRI sites or other appropriate sites. See, e.g.,FIG. 6 for a restriction map of the sequence of FIG. 1.

[0077] Dopamine receptor genes of this invention, particularly mammalianD₂ dopamine receptor genes, based on this disclosure, can now beroutinely made, isolated and/or cloned, using many conventionaltechniques. For example, the procedure disclosed herein can besubstantially reproduced for libraries containing dopamine receptor DNAsequences. Alternatively, oligonucleotide probes can be routinelydesigned, e.g., from the sequences of FIG. 1 and/or the omitted introns,which are selective for dopamine receptor genes, especially formammalian dopamine D₂ receptor genes. These can be used to screennucleic acid libraries containing dopamine receptor nucleic acidsequences. Sequences in these libraries hybridizing to the probes,especially to all of a plurality of such probes (e.g. 2 or 3), will beDNA sequences of this invention with high probability. Of course, it isalso possible to synthesize the sequence of FIG. 1 or any fragmentthereof using conventional methods.

[0078] This invention also enables the production of a wide variety ofuseful products and the employment of a wide variety of useful methods,as well as providing basic tools for the study of the regulation andfunction of dopamine receptors.

[0079] These products and methods can be produced and carried out,respectively, using the well known recombinant DNA, immunochemical andother methodologies of the biotech industry. See, e.g., U.S. Pat. No.4,237,224; U.S. Pat. No. 4,264,731; U.S. Pat. No. 4,273,875; U.S. Pat.No. 4,293,652; EP 093,619;

[0080] Davis et al “A Manual for Genetic Engineering, Advanced BacterialGenetics,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1980); Maniatis et al., Molecular Cloning, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; Davis et al., Basic Methods inMolecular Biology, Elsevier, N.Y. (1986); Methods in Enzymology, Berger& Kimmel (Eds.), (1987).

[0081] This invention provides, for the first time, purified andisolated polypeptide products having all or part of the primary aminoacid sequence of the dopamine D₂ receptor as well as its biologicalproperties, including: all sites for covalent modification, such asphosphorylation and glycosylation; all primary sequence that determinesthe secondary, tertiary and quaternary structure of the functionalprotein; all parts of the molecule that provide antigenicity andantibody binding sites; all parts of the protein that provide fornoncovalent interactions with other biological molecules, such aslipids, carbohydrates, and proteins, such as guanine nucleotide-bindingregulatory proteins; all parts of the protein that make up the bindingsites for ligands, including agonists, partial agonists and antagonists;all parts of the protein that are required for functional conformationalchanges involved in normal biological activities, such asdesensitization; and all proteins that might arise as a result ofalternative splicing of the gene for this receptor.

[0082] These polypeptides can be expressed from the nucleic acids ofthis invention by procaryotic or eucaryotic hosts, e.g., bacterial,yeast or mammalian cells in culture, using fully conventionaltransformation or transfection (e.g., via calcium phosphate formammalian cells) techniques. The products of such expression invertebrate (e.g., mammalian and avian) cells are especially advantageousin that they are produced free from association with other humanproteins or contaminants with which they may be associated in naturalform. Preferred hosts for expression are mammalian and include forexample mouse Ltk⁻cells, hamster CHO cells, mouse GH₄ cells, mouse C₆cells, mouse/rat NG108-15 cells and mouse AtT20 cells. For example, whenthe gene of FIG. 1 is transfected into the commercially available growthhormone GH₄ cells, modulation of the cAMP second messenger system hasbeen observed. Preferred vectors include pZem or pRSV or viral vectorssuch as vaccinia virus and retroviruses.

[0083] The polypeptides of this invention include all possible variants,e.g., both the glycosylated and non-glycosylated forms. The particularcarbohydrates involved will depend on the mammalian or other eucaryoticcells used for the expression. The polypeptides of the invention canalso include an initial methionine amino acid residue.

[0084] Also included in this invention are polypeptides, synthetic orotherwise, duplicating the amino acid sequence of FIG. 1 and/or of thedopamine receptors per se of this invention, or only partiallyduplicating the same. These wholly or partially duplicative polypeptideswill preferably also retain the biological and/or immunological activityof the dopamine receptor per se. Also included within the scope of thisinvention are the monoclonal and polyclonal antibodies (generatable byconventional techniques and preferably labelled) which areimmunoreactive with such polypeptides.

[0085] Preferred partial polypeptides (fragments) are those including atleast a portion of the sequences located in the hydrophobictransmembrane domains V, VI and VII, shown in FIG. 2. These are thelikely locations of the ligand binding site(s), particularly domain VII.The third cytoplasmic loop is also an important fragment area; e.g.,G-protein binding requires this location as well as domains V and VI.Where it is desired to have an antibody highly specific to a particulardopamine receptor, a fragment generating such an antibody will beselected from the highly unique region between transmembrane regions Vand VI, i.e., the third cytoplasmic loop, or the C-terminal domain, bothof which have low homology with other receptors, and/or the antibodieswill be selected to be specific to an epitope in these regions. Anotherreceptor/gene specific region is that of the intron sequences, e.g.,those for RGB-2, mentioned above. Particularly preferred peptides whichhave been synthesized are (referring to the amino acid numbers of FIG.1): (A) 2-13; (B) 182-192; (C) 264-277; (D) 287-298; and (E) 404-414.These are selected based on the following principles: the knownantigenicity of peptides containing a large number of Pro residues(B/C/D); coverage of the N and C termini (A) and (E); the ability todirect antibodies towards an extracellular domain (receptor reactionregion) which will be effective to block the receptor reaction (A/C/D).Antibodies to these fragments are raised conventionally, e.g.,monoclonals by fully conventional hybridoma techniques.

[0086] This invention also relates to DNA sequences encoding the fulldopamine receptor or fragments thereof, as well as expression vectors(e.g., viral and circular plasmid vectors) containing such whole orpartial sequences. Similarly, hosts (e.g., bacterial, yeast andmammalian) or cells transformed or transfected with such vectors arealso included. The corresponding methods of expressing the polypeptidescorresponding to the sequences in such vectors are also included, e.g.,comprising culturing the transformed or transfected cells underappropriate conditions for large scale expression of the exogenoussequences and for the isolation of the polypeptides as usual, e.g., fromthe growth medium, cellular lysates or cellular membrane fractions.

[0087] DNA sequences of this invention also include those coding forpolypeptide variants, mutants or analogues which differ from the naturalsequence described herein. Such differences can be derived from deletionof one or more amino acid residues from the natural sequence, bysubstitution of a given such residue by another residue and/or byadditions wherein one or more amino acid residues are added to thenatural sequence. Preferably, the resultant modified polypeptide willretain at least one of the biological or immunological activities of thedopamine receptor.

[0088] Mutations likely to affect dopamine affinity activity will bethose in transmembrane domains V, VI or VII or in the third cytoplasmicloop. In addition, the DNA sequences also include sequencescomplementary to any of the other DNA strands mentioned herein and, mostnotably, those shown in the Figures; DNA sequences which hybridize tothe DNA sequences described herein, typically under the hybridizationconditions mentioned herein or under more stringent conditions, or whichhybridize to fragments of such DNA sequences; and DNA sequences whichdiffer from those shown herein by the degeneracy of the genetic code.Thus, this invention includes all DNA sequences which encode a dopaminereceptor and hybridize to one or more of the sequences shown herein.These include allelic variants as well as dopamine receptors frommammalian species other than the species mentioned in the experimentaldescriptions herein.

[0089] Modifications of the cDNA or genomic dopamine receptor DNA may bereadily accomplished by any of the well known techniques, includingsite-directed mutagenesis techniques. Such modified DNA sequences caninclude deletions, additions and/or substitutions made in selectedregions, e.g., not in transmembrane domains V, VI or VII or in the thirdcytoplasmic loop, where retention of the underlying biological activityof the dopamine receptor is desired.

[0090] Modified proteins which do not retain the mentioned biologicalactivity and/or the corresponding DNA sequences will also be useful,e.g., in various assays of this invention. In a particularly preferredsuch modification, the transmembrane domain V, VI, or VII or the thirdcytoplasmic loop will be deleted or rendered inactive, e.g., by sequencemodification. Deletion of the glycosylation sites shown in FIG. 1 isalso a useful variant for expression of the polypeptide, e.g., in yeastcells.

[0091] As mentioned above, it is well established that significantportions of the DNA sequence encoding a dopamine receptor are conservedin various mammalian species. Consequently, using only routineexperimentation, a skilled worker can readily screen a DNA genomiclibrary or, preferably, a cDNA library, e.g., from the brain of a givenmammal, for the presence of other dopamine receptor genes, especially D₂dopamine receptor genes, using probes manufactured in accordance withthe details of the sequences shown herein, including the 5′ flanking,the intronic and the structural gene sequences shown in FIG. 1 and thehuman sequence of FIG. 7. Probes will preferably be selected from theseven highly conserved transmembrane domains shown in FIG. 2, preferablydomains VI and VII. Such a routine screening will identify clones whichhybridize with the probes. From these, dopamine receptors can routinelybe selected, e.g., using the techniques described herein. With respectto human D₂ dopamine receptors, particularly useful sources include, forcDNA, striatum, pituitary, neuroblastoma, kidney, placenta cells, etc.,and, for genomic DNA, liver, placenta cells, etc. For primates, e.g.,rhesus monkeys, particularly useful genomic DNA or cDNA librariesinclude brain, kidney and placenta cells.

[0092] With respect to human D₂ dopamine receptor genes, the partialsequence shown in FIG. 7 has been identified by conventionallyscreening, under the stringent hybridization conditions described abovefor the probing by the 0.8 kb EcoRI-PstI fragment of rat brain cDNA inλgt10, a pituitary cDNA library using a probe which is the full lengthrat cDNA, RGB-2. The cDNA libraries mentioned herein were prepared byfully conventional methods, e.g., as described in the references citedabove, e.g., Davis et al. This sequence or fragments thereof can also beuseful as a probe, for example, to screen conventional libraries asmentioned above for human dopamine receptor genes in accordance with theforegoing and other fully conventional procedures. As can be seen bycomparing the sequence of FIG. 7 with the sequences shown in FIGS. 1 and2 above, the partial human sequence of FIG. 7 has high homology withRGB-2 beginning at amino acid no. 259 of FIG. 1. similarly, thisinvention more generally includes mammalian D₂ dopamine receptor genesin the broadest sense, e.g., both regulatory and structural such genes,alone or in combination, e.g., in reading frame. For example, using theroutine methods discussed herein, such genes have been and can be clonedfrom mammalian DNA libraries. As well, this invention includesbiologically active fragments of such genes, e.g., fragments encodingpolypeptides having the biological activity of a mammalian D₂ dopaminereceptor, or fragments useful in controlling expression of such encodingfragments, or fragments useful as probes for any such gene or fragment,e.g., by hybridizing therewith.

[0093] The various polypeptides and sequences of this invention may beconventionally labeled with detectable marker substances, typically bycovalent association, and further typically by radiolabeling, or in thecase of DNA, with non-isotopic labels such as biotin. The polypeptideproducts (e.g., labelled antibodies) can be used conventionally todetect and quantitate the presence of dopamine receptors in varioussamples; the DNA-labeled products can be conventionally employed in theusual hybridization methods (e.g., Northern blots, Southern blots, spotassays, etc.) to detect and quantitate the presence of associatednucleic acids (DNA, RNA) in samples, e.g., to locate the dopamine genepositions in various mammalian chromosomal maps, to determine whethermRNA or receptor concentrations are abnormally high or low in comparisonto standard levels, etc. They will also be useful, again using fullyconventional procedures, to identify dopamine receptor gene disorders(defective or aberrant genes) in in vitro diagnostic procedures on DNAsamples from given patients, e.g., to detect chromosomal defects, e.g.,using RFLP analyses (see, e.g., Genes III, Levin, John Wiley and Sons(1987). For example, since dopamine receptors have been implicated inschizophrenia, products and methods of this invention can be used tocharacterize nucleic acids, e.g., in size fractionated form, fromschizophrenia patients and, inter alia, classify patients according toschizophrenia subgroups, make diagnoses based on comparisons to standardDNA fractionation arrays, etc. Of course, they can also be used, e.g.,in the mapping of the human or other mammalian genome, as gene markersto identify accompanying genes, e.g., in RFLP analyses, and, whereapplicable, other disorders. The gene-unique regions discussed above,e.g., the intron regions, the third cytoplasmic loop, etc., will beespecially useful in this regard.

[0094] Typical assays in which the polypeptides of this invention can beutilized include any of the well known immunoassay techniques such asRIA, ELISA, etc., both of in vitro and in vivo nature. Various fragmentsof the polypeptide sequence of the dopamine receptor can also beutilized conventionally for producing corresponding polyclonalantibodies or preferably monoclonal antibodies (e.g., by conventionalpreparation and expression of corresponding hybridomas) for epitopeswithin the given fragment. The antibodies will often be conventionallylabelled, e.g., radio- or enzymatically labelled. In a preferred aspect,the resultant polyclonal or monoclonal antibodies will also beimmunospecific with respect to not only the mentioned fragment, but alsothe full protein. Such antibodies will be conventionally employable, forexample, in the detection and affinity purification or chromatography ofdopamine receptor and related products.

[0095] Of course, the polypeptides of this invention include thoseexpressed in accordance with conventional procedures from cells asmentioned above, as well as those which are synthetically prepared alsousing conventional procedures. This invention enables for the first timenon-natural preparation of mammalian dopamine receptors substantiallyfree of constituents of their natural environment. It is in this sensethe term “substantially pure” is used herein, i.e., substantially freeof these natural constituents. The invention also includes DNA sequenceswhich can be isolated from the various sources mentioned above orsynthetically prepared using fully conventional methods.

[0096] Also included within the scope of this invention arepharmaceutical compositions comprising effective amounts of one or moreof the polypeptide products of this invention or one or more of thenucleic acid sequences of this invention, in admixture with suitableconventional diluents, adjuvants and/or carriers well known in thepharmaceutical industry. These can be utilized for in vitro uses, e.g.,for detection of the presence of a dopamine receptor in a sample or ofthe presence of a gene or an abnormal gene in a sample or for increasingthe concentration of receptor or its gene in a sample, or for in vivouses such as gene therapy (e.g., to render a defective gene or geneproduct inactive, e.g., block it by an appropriate monoclonal antibody)and/or to provide new functional genes (e.g., using retroviralvectors)), or to provide an increased concentration of dopamine receptorin a given location, or to modulate receptor expression and/or activity,e.g., by administration of antisense oligosequences, all in mammalsincluding humans. Thus, these compositions will be useful to treatdisease conditions, inter alia, those associated with abnormalities inthe structure, expression or concentration of the dopamine receptor orits gene, such as those mentioned in the foregoing. Specific effectivedosages for a given condition in a given patient will vary, as is wellknown, with the usual conditions, including the overall condition of thepatient, body weight, the identity and severity of the particulardopamine-deficiency disease state, etc.

[0097] The polypeptides of this invention can also be used in, e.g.,competitive binding assays, to test for the affinity thereto ofcandidate chemical substances such as drugs, e.g., affinity (e.g.,agonistic or antagonistic) to D₂ dopamine receptors. Such procedures canbe carried out, e.g., as pharmaceutical screening tests, using fullyconventional procedures, analogous to those described herein and/or toknown protocols based on natural sources of dopamine receptors, e.g.,analogous to known tests for inhibition of the binding of tritiateddopamine agonists and antagonists to striatal receptors per the methodsof Schwarcz et al., J. Neurochemistry, 34 (1980), 772-778 and Creese etal., European J. Pharmacol., 46 (1977), 377-381, and to those for otherreceptors. It is also possible to screen substances for ability tomodify or initiate a response which is triggered by ligand bonding to adopamine receptor, e.g., cellular responses such as modulation of secondmessenger systems. Such analyses can utilize cells of this inventiontransformed with nucleic acid sequences of this invention.

[0098] Antibodies of this invention to the dopamine receptors or otherregions of dopamine receptor genes, especially the D₂ receptor, can alsobe used in diagnostic imaging techniques, e.g., by radiodiagnostic, MRIor positron imaging. Radio-, paramagnetic- or positron-labels can beconventionally attached to the antibodies (preferably monoclonal innature), e.g., via covalent bonding to chelating groups for a positronemitting, radionucleotide or paramagnetic metal. MRI, radiosensitive orpositron imaging can then be effected with these agents usingconventional methods. See, e.g., Maziere et al., Life Sci., 35, 1349(1984).

[0099] Suitable pharmaceutical carriers include water, saline, humanserum albumin, etc. The compositions can also include other activeingredients suitable for amelioration of the particular disease stateinvolved, e.g., conventional dopamine agonists, dopamine antagonists,etc. The components of this invention can be provided in conventionalkit form containing, e.g., an antibody or a DNA probe (e.g., able todetect gene homologies or anomalies) along with detectionmethod-specific reagents such as enzymes, substrates, materials foranalyzing DNA restriction fragments, etc.

[0100] The DNA sequences of this invention are also useful to preparethe corresponding transgenic animals, in particular nonhuman mammals,e.g., rats, monkeys, etc., using known methods, e.g., analogous to thosedescribed in U.S. Pat. No. 4,736,866. Such animals, e.g., areparticularly useful for commercial research purposes. The DNA sequencesor the corresponding mRNA can also be used conventionally to injectoocytes, e.g., from frogs, which can then be conventionally used inbinding or second messenger analyses. Moreover, the availability of theprimary amino acid sequence itself enables experimental andcomputational modeling and understanding of the secondary and tertiarystructures of the dopamine receptor. This three-dimensional informationprovides a basis for modeling and understanding the details of thereceptor function, e.g., interaction with the cell membrane, its ligand(binding pocket), associated proteins, messenger systems, etc. Suchanalyses enable rational drug design whereby, e.g., new dopaminereceptor affecting chemical agents can be designed in accordance withthe details of this interaction.

NUMBERED REFERENCES FOR BACKGROUND, SUMMARY, FIGS. 1-7, AND DISCUSSION(EXCLUDING EXAMPLES)

[0101] 1. Creese, I., Sibley, D. R., Hamblin, M.W. & Leff, S. E. Ann.Rev. Neurosci. 6, 43-71 (1983).

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[0103] 3. Lee, T. et al. Nature 273, 59-61 (1984).

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[0105] 5. Barnes, D. M. Science 241, 415=417 (1988).

[0106] 6. Cote, T. E., Frey, E. A., Grewe, C. W., & Kebabian, J. W. J.Neural. Trans. Suppl. 18, 139-147 (1983).

[0107] 7. Senogles, S. E. et al. J. Biol. Chem. 262, 4860-4867 (1987).

[0108] 8. Dohlman, H. G., Caron, M. G. & Lefkowitz, R. J. Biochemistry26, 2657-2664 (1987).

[0109] 9. Dixon, R. A. F., et al. Nature 321, 75-79 (1986).

[0110] 10. Mount, S. M. Nucleic Acids Res. 10, 459-472 (1982).

[0111] 11. Grigoriadis, D. E., Niznik, H. B., Jarvie, K. R. & Seeman, P.FEBS Let. 227, 220-224 (1988).

[0112] 12. Kobilka, B. K., et al. Science 238, 650-656 (1987).

[0113] 13. Kobilka, B. K., et al. Nature 329, 75-79 (1987).

[0114] 14. Kubo, T., et al. Nature 323, 411-416 (1986).

[0115] 15. Kubo, Y., et al. Nature 329, 836-838 (1986).

[0116] 16. Strader, C. D., et al. J. Biol. Chem. 263, 10267-10271(1988).

[0117] 17. Sibley, D. R., Benovic, J. L., Caron, M. G. & Lefkowitz, R.J. Cell 48, 913-922 (1987).

[0118] 18. Bouvier, M., et al. Nature 333, 370-373 (1988).

[0119] 19. Boyson, S. J., McGonigle, P. & Molinoff, P. B. J. Neurosci.6, 3177-3188 (1986).

[0120] 20. Uhler, M. & McKnight, G. S. J. Biol. Chem. 262, 15202-15207(1987).

[0121] 21. Gorman, C., Padmanabhan, R. & Howard, B. H. Science 221,551-553 (1983).

[0122] 22. Hyttel, J. Eur. J. Pharmacol. 91, 153-154 (1983).

[0123] 23. Hamblin, M. W., Leff, S. E. & Creese, I. Biochem. Pharmacol.33, 872-877 (1984).

[0124] 24. Dolphin, A. C. Trends in Neurosci. 10, 53-57 (1987).

[0125] 25. Jones, S. V. P., et al. Proc. Nat. Acad. Sci. U.S.A. 85,4056-4060 (1988).

[0126] 26. Sanger, F., Nicklen, S. & Coulson, A. R. Proc. Nat. Acad.Sci. U.S.A. 74, 5463-5467 (1977).

[0127] 27. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J. & Rutter,W. J. Biochemistry 18, 5294-5299 (1979).

[0128] 28. Ullrich, A., et al. Science 196, 1313-1319 (1977).

[0129] 29. Neve, K. A. & Molinoff, P. B. Mol. Pharmacol. 30, 104-111(1986).

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[0131] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The mentioned embodiments are,therefore, to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever.

[0132] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

[0133] The entire texts of all applications, patents and publicationscited above are hereby incorporated by reference.

EXAMPLES Example 1

[0134] Summary

[0135] We recently cloned a complementary DNA for the rat dopamine D-2receptor, making it possible to create cell lines expressing thisreceptor. A cell line (LZR1) was created by transfecting the D-2 cDNA(RGB-2) into mouse fibroblast Ltk⁻ cells. LZR1 cells, previouslydescribed as L-RGB2Zem-1 cells (1), expressed a high density of D-2receptors, whereas the wild-type cells did not. Although transcriptionof the RGB-2 cDNA is regulated by the zinc-inducible mousemetallothionein promoter, the density of D-2 receptors on membranesprepared from LZR1 cells was not increased after the cells were treatedwith zinc. A second cell line derived from Ltk cells, designated LZR2,had a lower density of D-2 receptors in the uninduced state, andtreatment with zinc induced a 50% increase in the receptor density. Anumber of agonists competitively and stereoselectively inhibited thebinding of [³H]spiroperidol to the expressed D-2 receptors. In LZR1cells, dopamine was a more potent inhibitor of radioligand binding inthe absence than in the presence of GTP and NaCl. Dopamine reducedforskolin-stimulated adenylate cyclase activity by 27% in membranesprepared from LZR1 cells. Inhibition by dopamine was blocked by(+)-butaclamol or prior treatment of intact cells with pertussis toxin.These data indicate that the RGB-2 cDNA directs the expression of adopamine D-2 receptor capable of interacting with guaninenucleotide-binding proteins and inhibiting adenylate cyclase activity.Furthermore, the RGB-2 cDNA provides a means of creating many cell linesthat will be useful tools for the biochemical and pharmacologicalcharacterization of dopamine D-2 receptors.

[0136] Introduction

[0137] Dopamine (DA) receptors have been classified into two subtypesbased on functional and pharmacological profiles (2). DA D-2 receptorsare characterized functionally by their ability to inhibit adenylatecyclase activity (3). Activation of D-2 receptors also inhibits calciumchannels (4, 5), increases potassium conductance (6), and may inhibitaccumulation of inositol phosphates (7, 8). One factor that has impededresearch on the regulation and functional characteristics of DAreceptors has been the lack of cell lines that express the receptors.One cell line, derived from a prolactin-secreting tumor, has recentlybeen described in which DA inhibits adenylate cyclase activity andprolactin secretion (9).

[0138] We recently cloned a rat brain complementary DNA (cDNA),designated RGB-2, that has significant homology with β₂-adrenergicreceptors and other receptors that interact with guaninenucleotide-binding proteins. Three lines of evidence indicate that theRGB-2 cDNA encodes the DA D-2 receptor: (1) The deduced aminoacid-sequence of the protein suggests the existence of the sevenmembrane-spanning domains typical of receptors coupled to guaninenucleotide-binding proteins (10); (2) the distribution of messenger RNAthat hybridizes with the cDNA parallels the distribution of the D-2receptor; and (3) when the RGB-2 cDNA is transfected into cells thatlack high affinity binding of the D-2 selective ligand [³H]spiroperidol,the cells express binding sites for the radioligand with apharmacological profile characteristic of D-2 receptors (1).

[0139] The cloning of a D-2 receptor cDNA makes it possible to expressDA receptors in cell lines in which the effects of receptor activationcan readily be determined. We previously described the binding of[³H]spiroperidol and other D-2 antagonists to a line of cells derived bytransfection of mouse L cells with the RGB-2 cDNA under the control of azinc-inducible mouse metallothionein promoter (1). We also reportedthat, under the assay conditions used previously, the binding of DA toLZR1 membranes was not responsive to GTP. We now demonstrate that theD-2 receptor encoded by the RGB-2 cDNA is functional with respect to theguanine nucleotide sensitivity of the binding of agonists and theability of agonists to inhibit adenylate cyclase activity.

[0140] Methods

[0141] Materials: [α-³²P]Adenosine 5′-triphosphate (ATP, 10-50 Ci/mmol)and [³H]cyclic AMP (31.9 Ci/mmol) were purchased from New EnglandNuclear (Boston, Mass.), and [³H]spiroperidol (95 Ci/mmol) was purchasedfrom Amersham (Arlington Heights, Ill.). Guanosine 5′-triphosphate(GTP), DA, cyclic AMP, 3-isobutyl-1-methyl-xanthine, ATP, and forskolinwere purchased from Sigma Chemical Company (St. Louis, Mo.). Quinpirole,LY181990 (Lilly Laboratories), bromocriptine (Sandoz ResearchInstitute), and (+) and (−)3-PPP (Astra) were generous donations.

[0142] Transfection: The full RGB-2 cDNA was cloned into the plasmidpZem3 (11). The cDNA and the vector were made compatible by partiallyfilling in the Bg1 II site on the vector and a Sal I site on the cDNAadaptor. This plasmid was co-transfected with the plasmid pRSVneo intomouse Ltk⁻ cells by a CaPO₄ precipitation technique (12). Transfectantswere selected in 350 μg/ml of G418, isolated, and screened forexpression of RGB-2 mRNA by Northern blot analysis. The subclone LZR1,selected on the basis of high expression of RGB-2 mRNA, was partiallycharacterized previously as L-RGB2Zem-1 (1). A second cell line, LZR2,was isolated in the same way.

[0143] Tissue culture: Cells were plated at a density of 20,000cells/cm² in 150 mm diameter Falcon tissue culture plates (BecktonDickinson, Lincoln Park, N.J.), subcultured by replacing the growthmedium with trypsin-EDTA (0.1% trypsin, 0.02% EDTA in phosphate-bufferedsaline) or fed on day 3, and harvested on day 5 or 6. Cells were grownin Dulbecco's modified Eagles¹ medium (Sigma), supplemented with 5%fetal bovine serum and 5% iron-supplemented calf bovine serum (Hyclone,Logan, Utah), in an atmosphere of 10% CO₂/90% air at 37°. Cells werelysed by replacing the growth medium with ice-cold hypotonic buffer (1mM Na⁺-HEPES, pH 7.4, 2 mM EDTA). After swelling for 10-15 min, thecells were scraped from the plate and centrifuged at 24.000×g for 20min. The resulting crude membrane fraction was resuspended with aBrinkmann Polytron homogenizer at setting 6 for 10 sec in Tris-isosaline(50 mM Tris-HCl, pH 7.4, and 0.9% NaCl) and stored at −70° for receptorbinding experiments or resuspended in Tris-isosaline, centrifuged againat 24,000×g for 20 min., and resuspended in Tris-isosaline for immediateuse in adenylate cyclase experiments.

[0144] Receptor binding assay: The membrane preparation was thawed,centrifuged at 24,000×g for 20 min., and resuspended in Tris-isosalineexcept where indicated. Aliquots of the membrane preparation were addedto assay tubes containing (final concentrations) 50 mM Tris-HCl, pH 7.4,0.9% NaCl, 0.025% ascorbic acid, 0.001% bovine serum albumin,[³H]spiroperidol, and appropriate drugs. (+)-Butaclamol (2 μM) was usedto define nonspecific binding, which was typically less than 10% oftotal binding at concentrations of radioligand near the K_(D) value.Assays were carried out in duplicate in a volume of 2 ml for saturationanalyses or 1 ml for inhibition analyses. Incubations were initiated bythe addition of 15-50 μg of protein, carried out at 37° for 50 min., andstopped by the addition of 10 ml of ice-cold wash buffer (10 mM Tris, pH7.4, and 0.9% NaCl) to each assay. The samples were filtered throughglass-fibre filters (Schleicher & Schuell No. 30) and washed with anadditional 10 ml of wash buffer. The radioactivity retained on thefilters was counted using a Beckman LS 1701 scintillation counter. Datawere analyzed by nonlinear regression using the data analysis programEnzfitter (Elsevier-Biosoft). In competition experiments, K_(I) valueswere calculated from experimentally determined IC₅₀ values by the methodof Cheng and Prusoff (13). Averages for K_(I) and K_(D) values are thegeometric means. In experiments designed to assess the effect of GTP andNaCl on the binding of DA, fresh tissue was used. Cells were harvested,centrifuged, and Mg²⁺ resuspended in Tris-Mg²⁺ (50 mM Tris, pH 7.4, and4 mM MgCl₂). Tissue was incubated for 15 min. at 37° in this bufferbefore re-centrifugation. The resuspended protein was added to assayscontaining Tris-Mg²⁺ with no added NaCl or GTP, or Tris-Mg²⁺ with 120 mMNaCl and 100 μM GTP.

[0145] Adenylate cyclase assay: The conversion of [α³²P]ATP to [³²P]cAMPwas determined essentially as described by Salomon et al. (14).Membranes (50-100 μg of protein) resuspended in Tris-isosaline wereadded in a volume of 0.1 ml to an assay of 0.2 ml containing 50 mMTris-HCl, pH 7.4, 5 mM cAMP, 1 mM 3-isobutyl-1-methylxanthine 1 mMMgCl₂, 0.5 mM EGTA, 0.25 mM ATP, 30 μM GTP, approximately 2×10⁶ cpm of[α-³²P]ATP, and various drugs. Assays were initiated by warming to 25°and terminated after 20 minutes by cooling to 0°, then addingtrichloroacetic acid (100 μl of a 30% solution) to each assay.[³H]Cyclic AMP (approximately 30,000 cpm) was added to each assay as aninternal standard. The assay volume was brought up to 1 ml with water,and tubes were centrifuged for 10 min. at 2000×g. Cyclic AMP in thesupernatant was isolated by sequential chromatography on columnscontaining Dowex AG50W-X4 resin and neutral alumina. The 2-ml eluatefrom each column of alumina was dissolved in 10 ml of Bio-Safe II (RPI,Mount Prospect, Ill.) for liquid scintillation counting. Dose-responsecurves for inhibition of adenylate cyclase activity by agonists wereanalyzed by nonlinear regression using the program Enzfitter. The datawere fit to the equation:

E=(100−E _(max))/(1+(A/EC ₅₀)^(N))+E _(max)

[0146] where E is the amount of enzyme activity, expressed as percentageof total stimulated activity, A is the concentration of agonists, EC₅₀is the concentration of agonist causing half-maximal inhibition ofenzyme activity, E_(max) is the enzyme activity observed in the presenceof maximally inhibiting concentrations of agonist, expressed as thepercentage of total stimulated activity, and N is a slope factor.Averages of EC₅₀. values are the geometric means. Protein concentrationwas determined by the method of Peterson (15).

[0147] Results

[0148] Saturation analysis of the binding of [³H]spiroperidol: Thedensity of D-2 receptors on membranes prepared from LZR1 cells wasdetermined by saturation analysis of the binding of [³H]spiroperidol(FIG. 8). Since the RGB-2 cDNA is under the control of thezinc-inducible mouse metallothionein promoter, the effect of priortreatment of cells with 100 μM zinc sulfate on the binding of[³H]spiroperidol was also determined. The density of binding sites was736±140 fmol/mg of protein in control LZR1 cells, and 759±155 fmol/mg ofprotein in LZR1 cells treated with zinc (n=2). A second cell line,designated LZR2, had a lower density of D-2 receptors (mean B_(max)±SE,435±71 fmol/mg of protein). In contrast to LZR1 cells, the density ofbinding sites on LZR2 cells was increased 50% by zinc treatment to630±52 fmol/mg of protein. The mean K_(D) value of 42 pM for controlLZR1 and LZR2 cells (pK_(D)±SE, 10.37±0.15, n=4) did not differsignificantly from the mean K_(D) value of 47 pM for zinc-treated cells(10.33±0.17). Scatchard transformation of saturation analyses from allexperiments yielded straight lines. Wild-type Ltk⁻ cells had nodetectable displaceable binding of [³H]spiroperidol (data not shown).Since LZR1 cells had the higher density of D-2 receptors, these cellswere used in all subsequent experiments.

[0149] Inhibition of radioligand binding by agonists: The apparentaffinity of D-2 receptors for several agonists and related compounds wasdetermined in two experiments (FIG. 9A, Table 1). Of the six compoundstested, bromocriptine was the most potent with a mean K_(I) value of 2nM, whereas LY181990, the inactive enantiomer of the D-2-selectiveagonist quinpirole, was the least potent. All assays were carried out inthe presence of 0.1 mM GTP and 120 mM NaCl, using membranes preparedfrom LZR1 cells.

[0150] In other experiments, the effect of GTP and NaCl on inhibition ofradioligand binding by DA was determined (FIG. 9B). Freshly preparedmembranes were used for these experiments, and 4 mM MgCl₂ was added tothe homogenization and assay buffers. In the absence of GTP and NaCl,the mean IC₅₀ was 25 μM [mean−log(IC₅₀)±SE=4.6±0.3]. Addition of GTP andNaCl to the assay buffer increased the mean IC₅₀ value to 167 μM(3.8±0.3, n=0.4), without altering the binding of [3H]spiroperidol. Hillcoefficients in the absence of GTP and NaCl ranged from 0.56 to 0.68(mean=0.64) whereas in the presence of GTP and NaCl values ranged from0.77 to 1.1 (mean=0.94). In the absence of GTP and NaCl, inhibitioncurves for DA could be fit best by assuming the presence of two classesof binding sites. One class of high affinity sites, representing 48±14%of the total number of receptors, had a mean K_(I) for DA of 0.3 μM. Thesecond class, representing 52±13% of the total number of receptors had amean K_(I) value of 24 μM.

[0151] Inhibition of adenylate cyclase activity by agonists: In freshlyprepared membranes from LZR1 cells, but not in membranes from wild-typeLtk⁻ cells, DA caused a concentration-dependent attenuation offorskolin-stimulated adenylate cyclase activity. Maximal inhibition was27% of total activity, with an EC₅₀ value of 624 nM (FIG. 10A; Table 1).Quinpirole was approximately as efficacious as dopamine; that is, themaximal inhibition induced by quinpirole was similar to that induced byDA. LY181990 was less potent and less efficacious than its active isomer(FIG. 10B; Table 1), indicating that D-2 receptor-mediated inhibition ofadenylate cyclase activity was stereoselective. The finding thatLY181990 caused measurable inhibition of enzyme activity in only 2 outof 3 experiments (Table 1) suggests that the compound has little or noagonist activity. Similarly, (−)3-PPP did not have detectable agonistactivity. Bromocriptine, with an EC₅₀ value of 45 nM, was the mostpotent agonist. Bromocriptine and (+)3-PPP were less efficacious thanDA; thus, the drugs appeared to be partial agonists.

[0152] Inhibition of adenylate cyclase activity in LZR1 cells by 10 μMDA was prevented by including 10 μM (+)-butaclamol in the assay (FIG.11), indicating that inhibition of DA is receptor-mediated. Also,treatment of LZR1 cells with pertussis toxin (50 ng/ml of growth mediumfor 16 hours) blocked DA-inhibited enzyme activity in membranes preparedfrom the cells (FIG. 10C), suggesting that G_(i) mediates inhibition ofenzyme activity by DA. Interestingly, forskolin-stimulated adenylatecyclase activity in membranes from pertussis toxin-treated cells wasapproximately 2.5-fold greater than activity in control membranes. TABLE1 Inhibition of radioligand binding and adenylate cyclase activity byagonists The apparent affinity (K₁) of 6 drugs for D-2 receptors onmembranes prepared from LZR1 cells, determined by inhibition of thebinding of [³H]spiroperidol (0.2 nM), is shown, as well as theconcentration of each drug that caused half-maximal inhibition offorskolin-stimulated adenylate cyclase activity (EC₅₀). Values for drugconcentrations, expressed as μM, are the geometric means of results from3 experiments (EC₅₀, quinpirole and (+) 3-PPP), 4 experiments (EC₅₀-DA)or 2 (K₁, all drugs; EC₅₀, bromocriptine) experiments. The maximalinhibition of adenylate cyclase activity observed (Max) is expressed asthe mean ± SEM of the percent inhibition of total activity in thepresence of 10 μM forskolin. For LY181990, the results shown are fromtwo experiments in which inhibition of enzyme was observed. There was noinhibition in a third experiment. Drug K₁ EC₅₀ Max Dopamine 17 0.6 27 ±3% Quinpirole 9 0.7 28 ± 2% LY181990 277 5.0 10 ± 2% Bromocriptine0.0024  0.04 17 ± 1% (+) 3-PPP 33 4.0 16 ± 3% (−) 3-PPP 0.87 — —

[0153] Discussion

[0154] As reported previously, Ltk⁻ cells transfected with a rat D-2receptor cDNA express a high density of DA D-2 receptors (1). We havecharacterized one subclone of these cells, designated LZR1, that stablyexpresses D-2 receptors at a density of 750 to 1000 fmol/mg of protein(present results; ref. 1).

[0155] As the D-2 cDNA is contained in the plasmid pZem3, under theregulation of a zinc-inducible promoter, the effect of zinc treatment onthe properties of radioligand binding was determined. D-2 receptors onLZR1 cells appear to be maximally expressed in the absence of zinc, sothat zinc treatment caused no increase in the density of receptors. Thelack of responsiveness to zinc is not a characteristic of the LtK⁻ cellline, since we have isolated a second transfected line of Ltk⁻ cells,LZR2, in which the density of D-2 receptors is elevated approximately50% by treatment with zinc, from 435 to 630 fmol/mg of protein. Theaffinity of D-2 receptors for [³H]spiroperidol was not significantlyaltered by zinc treatment.

[0156] The apparent affinity of D-2 receptors on LZR1 cells for severalagonists and related drugs was determined by inhibiting the specificbinding of [3H]spiroperidol with the drugs in the presence of GTP.Bromocriptine was an extremely potent agonist, with a K_(I) value of 2nM. The potency of quinpirole, like that of DA, was approximately 10 μM.The binding of agonists was stereoselective, since LY181990 was muchless potent than its active enantiomer, quinpirole, and (−)3-PPP wasmore potent than (+)3-PPP. These data extend our previous investigationof the binding of antagonists to D-2 receptors on LZR1 cells (1).

[0157] Physiological effects of stimulation of D-2 receptors appear tobe mediated by a guanine nucleotide-binding protein, G_(i), thatinhibits adenylate cyclase activity (16). High affinity binding ofagonists is thought to represent a ternary complex composed of agonist,receptor, and the α-subunit of G_(i) (G_(iα)), and inhibition of agonistbinding to D-2 receptors by GTP represents GTP-induced uncoupling of D-2receptors from G_(iα). To evaluate the ability of D-2 receptors encodedby the RGB-2 cDNA to couple to G proteins, the effect of GTP on thepotency of DA for inhibition of radioligand binding was determined. Inpreliminary studies using LZR1 cells, we found that in our standardassay buffer containing 120 mM NaCl and no added Mg²⁺, the binding of DAwas not sensitive to GTP (1), although under the same conditions thebinding of DA to rat striatal membranes is inhibited by GTP (data notshown). There were three possible explanations for the lack ofsensitivity to GTP in LZR1 membranes: (1) LZR1 cells, derived from Ltk⁻cells, could lack the appropriate G-protein. (2) The RGB-2 cDNA couldencode only a binding subunit of the D-2 receptor. This possibilityseemed unlikely because of the similarity between the predicted primarystructure of the protein encoded by RGB-2 and other receptors coupled toguanine nucleotide-binding proteins. (3) It could be that the ionicconditions of the binding assay were not appropriate for formation ordestabilization of the ternary complex. In the studies described here,ionic conditions were varied to increase the likelihood of observingGTP-sensitive binding. To maximize high-affinity agonist binding, tissuewas preincubated with MgCl₂, and MgCl₂ was included in the assay buffer(17). To maximize GTP-induced destabilization of the ternary complex,NaCl was added together with GTP (17). Under these conditions, additionof GTP and NaCl decreased the potency of DA for D-2 receptors andincreased the slope of the inhibition curve in membranes from LZR1cells. It is interesting that the ionic requirements for formation anddestabilization of the ternary complex seem-to be more stringent inmembranes from LZR cells than in membranes from rat striatum.

[0158] Inhibition of adenylate cyclase activity by DA D-2 receptors is awell-characterized phenomenon (3, 18, 19). Inhibition of adenylatecyclase activity by several drugs was assessed in membranes from LZR1cells. Maximally effective concentrations of DA and the D-2-selectiveagonist quinpirole decreased forskolin-stimulated enzyme activity byalmost 30%. Inhibition of enzyme activity by DA was blocked by the D-2antagonist (+)-butaclamol. The most potent agonist tested,bromocriptine, appeared to be a partial agonist, as reported by others(19, 20). Inhibition of adenylate cyclase by agonists wasstereoselective, since LY181990, the dextrorotatory enantiomer ofquinpirole, had little or no efficacy. As has been reported previously,the partial agonist (+)3-PPP is a stronger agonist than (−)3-PPP,although (−)3-PPP binds to D-2 receptors with higher affinity (21, 22).We observed no inhibition of adenylate cyclase activity by (−)3-PPP inmembranes from LZR1 cells. The EC₅₀ values determined for inhibition ofadenylate cyclase activity by agonists were generally lower than theK_(I) values determined in assays of ligand binding (Table 1). Thiscould be due to the presence of a receptor reserve on these cells,although the observation that drugs differed in maximal inhibition ofenzyme activity suggests that there is not a large receptor reserve evenfor the most efficacious agonists, DA and quinpirole. Also, there wouldbe no receptor reserve for a partial agonist such as (+)3-PPP. Analternative explanation is that inhibition of adenylate cyclase activitycould be related to the binding of agonists to D-2 receptors in ahigh-affinity state induced-by formation of the ternary complex, as hadbeen proposed for inhibition of enzyme activity by DA in the anteriorpituitary (23). On the other hand, K_(I) values in the present report,determined in the presence of GTP and NaCl, reflect the binding ofagonists to receptors in a state of low affinity. Direct comparisons aredifficult, since adenylate cyclase and radioligand binding assays werecarried out at different temperatures, but the K_(I) value for DAbinding to the high-affinity class of sites (0.3 μM) is close to theEC₅₀ value for DA-inhibited adenylate cyclase (0.6 μM), whereas theK_(I) values for the binding of DA to the low-affinity class of sites(24 μM) and binding in the presence of GTP (17 μM) are considerablyhigher.

[0159] DA did not inhibit adenylate cyclase activity in membranes fromLZR1 cells that had been treated with pertussis toxin. Since pertussistoxin-catalyzed ADP-ribosylation of G_(i) prevents G_(i)-mediatedinhibition of adenylate cyclase, this finding is consistent with thehypothesis that D-2 receptors interact with G_(iα) in the transfectedLZR1 cells. As has been observed for stimulation of adenylate cyclaseactivity by isoproterenol after pertussis toxin-treatment of other celltypes (24), treatment of intact LZR1 cells with pertussis toxinpotentiated the ability of forskolin to stimulate adenylate cyclaseactivity, suggesting that in some cell lines G_(i) normally acts toattenuate forskolin-and hormone-stimulated adenylate cyclase activity.

[0160] We have characterized a cell line, transfected with the RGB-2cDNA, that stably expresses a high density of D-2 receptors. With thiscell line, it was determined the cDNA encodes a DA D-2 receptor thatinteracts productively with a guanine nucleotide-binding protein toinhibit adenylate cyclase activity. It seems likely that the RGB-2 cDNAwould direct the expression of a functional D-2 receptor in almost anytype of cell. For example, GH₄C₁ cells, derived from a rat pituitarytumor (25), are prolactin-secreting cells that lack DA receptors, eventhough lactrotrophs in the rat anterior pituitary express D-2 receptors.Transfection of the RGB-2 cDNA into GH₄C₁ cells results in theexpression of a D-2 receptor with functional characteristics similar tothose described here (26). Cell lines created by transfection with a D-2receptor cDNA will be useful in the study of mechanisms of action andregulation of D-2 receptors.

REFERENCES FOR EXAMPLE 1

[0161] 1. Bunzow, J. R., H. H. M. Van Tol, D. K. Grandy, P. Albert, J.Salon, M. Christie, C. A. Machida, K. A. Neve, and O. Civelli. Cloningand expressing of a rat D₂ dopamine receptor cDNA. Nature (Lond.)336:783-787 (1988). 2. Kebabian, J. W., and D. B. Calne. Multiplereceptors for dopamine. Nature (Lond.) 277:93-96 (1979).

[0162] 3. De Camilli, P., D. Macconi, and A. Spada. Dopamine inhibitsadenylate cyclase in human prolactin-secreting pituitary adenomas.Nature (Lond.) 278:252-254 (179).

[0163] 4. Malgaroli, A., L. Vallar, F. R. Elahi, T. Pozzan, A. Spada,and J. Meldoiesi. Dopamine inhibits cytosolic Ca²-increases in ratlactotroph cells: Evidence of a dual mechanism of action. J. Biol. Chem.262:13920-13927 (1987).

[0164] 5. Drouva, S. V., E. Rerat, C. Bihoreau, E. Laplante, R.Rasolonjanahary, H. Clauser, and C. Kordon. Dihydropyridine-sensitivecalcium channel activity related to prolactin, growth hormone, andluteinizing hormone release from anterior pituitary cells in culture.Interactions with somatostatin, dopamine, and estrogens. Endocrinology123:2762-2773 (1988).

[0165] 6. Lacey, M. G., N. B. Mercuri, and R. A. North. Dopamine acts onD2 receptors to increase potassium conductance in neurones of the rabsubstantia nigra zona compacta. J. Physiol. (Lond.) 392:397-416 (1987).

[0166] 7. Simmonds, S. H., and P. G. Strange. Inhibition of inositolphospholipid breakdown by D₂ dopamine receptors in dissociated bovineanterior pituitary cells. Neurosci. Lett. 60:267-272 (1985).

[0167] 8. Enjalbert, A., F. Sladaczek, G. Guillon, P. Bertrand, C. Shu,J. Epelbaum, A. Garcia-Sainz, S. Jard, C. Lombard, C. Kordon, and J.Bockaert. Angiotensin II and dopamine modulate both cAMP and inositolphosphate productions in anterior pituitary cells: Involvement inprolactin secretion. J. Biol Chem. 261:4071-4075 (1986).

[0168] 9. Judd, A. M., I. S. Login, K. Kovacs, P. C. Ross, B. L.Spangelo, W. D. Jarvis, and R. M. MacLeod. Characterization of the MMQcell, a prolactin-secreting cloned cell line that is responsive todopamine. Endocrinology 123:2341-2350 (1988).

[0169] 10. Lefkowitz, R. J. and M. G. Caron. Adrenergic receptors:Models for the study of receptors coupled to guanine nucleotideregulatory proteins. J. Biol. Chem. 263:4993-4996 (1988).

[0170] 11. Uhler, M. D., and G. S. McKnight. Expression of cDNAs for twoisoforms of the catalytic subunit of cAMP-dependent protein kinase. J.Biol. Chem. 262:15202-15207, 1987.

[0171] 12. Gorman, C., R. Padmanabhan, and B. H. Howard. High efficiencyDNA-mediated transformation of primate cells. Science 231:551-553(1983).

[0172] 13. Cheng, Y. -C. and W. H. Prusoff. Relationship between theinhibition constant (K_(I)) and the concentration of inhibitor whichcauses 50 percent inhibition (I₅₀) of an enzymatic reaction. Biochem.Pharmacol. 22:3099-3108 (1973).

[0173] 14. Salomon, Y., C. Londos, and M. Rodbell. A highly sensitiveadenylate cyclase assay. Analyt. Biochem. 58:541-548 (1974).

[0174] 15. Peterson, G. L. A simplification of the protein assay methodof Lowry et al. which is more generally applicable. Analyt. Biochem.83:346-356 (1977).

[0175] 16. Cote, T. E., E. A. Frey, C. W. Grewe, and J. W. Kebabian.Evidence that the dopamine receptor in the intermediate lobe of the ratpituitary gland is associated with an inhibitory guanyl nucleotidecomponent. J. Neural. Trans. Suppl. 18:139-147 (1983).

[0176] 17. Hamblin, M. W., and I. Creese. ³H-Dopamine binding to ratseriatal D-2 and D-3 sites: Enhancement by magnesium and inhibition byguanine nucleotides and sodium. Life Sci. 30:1587-1595 (1982).

[0177] 18. Weiss, S., M. Sebben, J. A. Garcia-Sainz, and J. Bockaert.D₂-Dopamine receptor-mediated inhibition of cyclic AMP formation instriatal neurons in primary culture. Mol. Pharmacol. 27:595-599 (1985).

[0178] 19. Onali, P., M. C. Olianas, and G. L. Gessa. Characterizationof dopamine receptors mediating inhibition of adenylate cyclase activityin rat striatum. Mol. Pharmacol. 28:138-145 (1985).

[0179] 20. Agui, T., N. Amlaiky, M. G. Caron, and J. W. Kebabian.Binding of [¹²⁵I]-N-(p-aminophenethyl)spiroperidol to the D-2 dopaminereceptor in the neurointermediate lob of the rat pituitary gland: Athermodynamic study. Mol. Pharmacol. 32:163-169 (1988).

[0180] 21. Koch, S. W., B. K. Koe, and N. G. Bacopoulos. Differentialeffects of the enantiomers of 3-(3-hydroxyphenyl)-N-n-propylpiperidine(3-PPP) at dopamine receptor sites. Eur. J. Pharmacol. 92:279-283(1983).

[0181] 22. Meller, E., K. Bohmaker, Y. Namba, A. J. Friedhoff, and M.Goldstein. Relationship between receptor occupancy and response atstriatal dopamine autoreceptors. Mol. Pharmacol. 31:592-598 (1987).

[0182] 23. Borgundvaag, V., and S. R. George. Dopamine inhibition ofanterior pituitary adenylate cyclase is mediated through thehigh-affinity state of the D₂ receptor. Life Sci. 37:379-386 (1985).

[0183] 24. Abramson, S. N., M. W. Martin, A. R. Hughes, T. K. Harden, K.A. Neve, D. A. Barrett, and P. B. Molinoff. Interaction of β-adrenergicreceptors with the inhibitory guanine nucleotide-binding protein ofadenylate cyclase in membranes prepared from cyc-S49 lymphoma cells.Biochem. Pharmacol. 37:4289-4297 (1988).

[0184] 25. Tashjian, A. H. Clonal strains of hormone-producing pituitarycells. Meth. Enzymol. 58:527-535 (1979).

[0185] 26. Albert, P. R., K. Neve, J. Bunzow, and O. Civelli. Biologicalfunctions of the rat dopamine D₂ receptor cDNA expressed in GH₄C₁ ratpituitary cells. Proc. Endocrine Soc. 71:(in press).

Example 2

[0186] Summary

[0187] We have previously described a cDNA which encodes a binding sitewith the pharmacology of the D₂-dopamine receptor (Bunzow, J. R., et al.(19868) Nature 336, 783-787). We demonstrate here that this protein is afunctional receptor, i.e., it couples to G-proteins to inhibit cAMPgeneration and hormone secretion. The cDNA was expressed in GH₄C₁ cells,a rat somatomammotrophic cell strain which lacks dopamine receptors.Stable transfectants were isolated and one clone, GH₄ZR₇, which had thehighest levels of D₂-dopamine receptor mRNA on Northern blot, wasstudied in detail. Binding of D2-dopamine antagonist ³H-spiperone tomembranes isolated from GH₄ZR₇ cells was saturable, with K_(D)=96 pM,and B_(max)=2300 fmol/mg protein. Addition of GTP/NaCl increased theIC₅₀ value for dopamine competition for ³H-spiperone binding bytwo-fold, indicating that the D₂-dopamine receptor interacts with one ormore G proteins. To assess the function of the dopamine binding site,acute biological actions of dopamine were characterized in GH₄ZR₇ cells.Dopamine decreased resting intra- and extracellular cAMP levels by50-70% (EC₅₀=8±2 nM), and blocked completely VIP-induced enhancement ofcAMP levels (EC₅₀=6±1 nM), which ranged from 8-12 times basal levels.Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels byspiperone, (+)-butaclamol, (−)-sulpiride and SCH23390 occurred atconcentrations expected from K_(I) values for these antagonists at theD₂-receptor and was stereo-selective. Dopamine (as well as severalD₂-selective agonists) inhibited forskolin-stimulated adenylate cyclaseactivity by 45±6%, with EC₅₀ of 500-800 nM in GH₄ZR₇ membranes.Dopaminergic inhibition of cellular cAMP levels and of adenylate cyclaseactivity in membrane preparations was abolished by pretreatment withpertussis toxin (50 ng/ml, 16h). Dopamine (200 nM) abolished VIP andTRH-induced acute prolactin release. These data show conclusively thatthe cDNA clone encodes a functional dopamine-D2 receptor which couplesto G proteins to inhibit adenylate cyclase, and both cAMP-dependent andcAMP-independent hormone secretion. The GH₄ZR₇ cells will prove usefulin elucidating further the biochemistry of the dopamine D₂ receptor.

[0188] Introduction

[0189] The major element controlling PRL¹ secretion from the pituitaryis the concentration of dopamine in the hypophyseal portal bloodstream(1) Dopamine acts via dopamine-D₂ receptors on pituitary lactotrophs toinhibit basal and hormone-stimulated secretion of PRL (1-5). Thedopamine-D₂ receptor interacts with pertussis toxin-sensitive,inhibitory G proteins (6-9) to reduce adenylate cyclase activity, and toblock enhancement of cAMP levels by other agents (6, 10-12). Dopaminealso decreases [Ca⁺⁺]_(i) in lactotrophs, and partially inhibitselevation of [Ca⁺⁺]_(i) by other agents, such as TRH (13-15). Bothdopaminergic inhibition of cAMP and of [Ca⁺⁺]_(i) are mediated throughcoupling to one or more pertussis toxin-sensitive G proteins, and appearto contribute to dopamine inhibition of PRL secretion (15). The preciserelation between these components of dopamine action has been difficultto study (15, 16) due to the presence of heterogeneous cell types,limitations of cell number, and variations in responsiveness of diverslactotroph preparations.

[0190] The recent cloning of the dopamine-D₂ receptor cDNA (17) providesa useful tool to examine the intracellular actions and regulation of thereceptor. To examine whether the D₂-receptor clone directs synthesis ofa functional receptor, and to define the pathway between dopamine-D₂receptor activation and biological effect, we have transfected thedopamine-D₂ receptor cDNA into a pituitary-derived cell strain, GH₄C₁cells. GH₄C₁ cells are rat pituitary cells which synthesize and secretePRL and GH, and possess a variety of hormone, growth factor, andneurotransmitter receptors, second messenger systems, and ion channelsand have provided an accessible model of lactotroph function (18, 19).However, these cells lack dopamine-D₂ receptors, which are present onnormal lactotrophs, and thus provide an ideal host for studying thefunction of the dopamine-D₂ receptor. This report demonstrates that thegene product of the cDNA clone functions as a dopamine-D₂ receptor andcouples to inhibitory G proteins to decrease cAMP accumulation² and PRLrelease. The GH₄C₁ transfectants characterized herein should provide auseful cell system in which the mechanisms of dopamine action at D₂receptors may be studied further.

[0191] Experimental Procedures

[0192] Materials: Dopamine agonists and antagonists were from ResearchBiochemicals Incorporated (Waltham, Mass.), except quinpirole (Lilly),bromocryptine (Sandoz Research Institute), and (+) and (−)3-PPP (Astra).Rabbit antibody (lot CA-3) to 2-O-succinyl-cAMP-bovine serum albuminconjugate was obtained from ICN (Irvine, Calif.), rPRL standard andanti-rPRL antibody were from Dr. Salvatore Raiti, NIDDK, Bethesda, Md.Peptides were from Peninsula (, CA) or Sigma (St. Louis, Mo). α³²P-dCTP(2,200 Ci/mmol), ¹²⁵I-2-O-(iodotyrosyl methyl ester)-succinyl cAMP(2,200 Ci/mmol), ¹²⁵I-rPRL (2,200 Ci/mmol), α³² P-ATP (10-50 Ci/mmol),³H-cAMP (31.9 Ci/mmol) were from New England Nuclear (Boston, Mass.).All other chemicals were reagent grade, obtained primarily from Sigma.

[0193] Methods:

[0194] Construction of pZEM-D₂-cDNA: The pZEM-3 plasmid (20) was cut atthe Bgl II site between the metallothionein promotor and hGH 3′-flankingsequence. Full-length dopamine-D₂ cDNA (17) was excised from λGT10 withSal I and was ligated to the cut pZEM-3 plasmid in the presence of dATPand dGTP (250 μM) and transformed into E. coli strain XL-1 (Stratagene).Recombinants were characterized by their hybridization to ³²P-labelledD₂-cDNA, followed by restriction analysis and DNA sequencing.Recombinants with cDNA inserts in the sense orientation were preparedand purified by CsCl gradient centrifugation for transfection intoeucaryotic cells.

[0195] Cell Culture: GH₄C₁ cells, obtained from Dr. A. H. Tashjian, Jr.(Harvard University, Boston, Mass.) and subclones were grown in Ham'sF10 medium, supplemented with 10% fetal bovine serum, at 37° C. in 5%CO₂. For studies of ³H-spiperone binding or adenylate cyclase activity,cells were grown in Dulbecco's modified Eagle's medium supplemented with10t fetal bovine serum at 37° C. in 10% Co₂. Media were changed 12-24 hprior to transfection or experimentation. For transfection, GH₄C₁ cellswere grown to 2-4×10⁶ cells/10 cm dish. 20 μg of pZEM-D₂cDNA and 1 μgpRSV-neo were co-precipitated with calcium phosphate in 2 ml ofHepes-buffered saline, and placed over cells for 10-20 min. 8 ml of warmF10+10% fetal calf serum (pH 7.0) was added, and the cells incubated for4-5 h, 37° C. The medium was removed and 15% glycerol in Hepes-bufferedsaline was added, and incubated for 3 min., 37° C. The plates wererinsed and fresh F10+10% fetal calf serum added, and the cells placed inthe incubator for 16-20 h. Fresh medium supplemented with 700 μg/ml G418(Geneticin, GIBCO, N.Y.) was added over the next 3-4 weeks, to selectfor stable transfectants expressing neo-resistance. Single colonies wereisolated using sterile micropipette tips to take up individual coloniesin 3-5 μl. Once stocks of the transfectant cell lines were stored frozenin liquid nitrogen, G418 was omitted from growth media.

[0196] RNA Isolation and Northern Blot Analysis: Cells were rinsed incalcium-free Hepes-buffered saline+0.02% EDTA and extracted withTris-buffered guanidinium hydrochloride, centrifuged (33,000 rpm, 16 h)through a 1.7 g/ml CsCl pad, and the pellets extracted withphenol/chloroform and ethanol precipitated (21). RNA was resuspended andquantitated by UV absorbance at OD=260 nm. For Northern blots, RNA wasdenatured in glyoxal/dimethylsulfoxide (1 h, 50° C.), and run on a 1%agarose gel in 10 mM sodium phosphate. RNA was blotted overnight ontonylon membrane (N-bond, Amersham), baked at 80° C. for 2h.Prehybridization was as described, for 6 h at 42° C. Random-primed³²P-labelled 1.6 kb BamHI-BglII fragment of the D₂-cDNA (1-2×10⁶ dpm/μg)was used for hybridization, 16-20h at 42° C. in 50% formamide. Blotswere washed in 2×SSC for 10 min, room temperature, followed by 3×15 min.wash in 0.2×SSC, 0.5% SDS, 70° C., and exposed to X-ray film overnightat −80° C., with intensifying screen.

[0197] Ligand Binding: Cell membranes were prepared by first replacinggrowth medium with ice-cold hypotonic buffer (1 mM Hepes, pH 7.4, 2 mMEDTA). After swelling for 10-15 min, the cells were scraped from theplate and centrifuged at 24,000 g for 20 min, lysed with a BrinkmanPolytron homogenizer at setting 6 for 10 sec in Tris-isosaline (50 mMTris, pH 7.4, 0.9% NaCl) and stored at −70° C. for receptor bindingexperiments, or resuspended in 50 mM Tris, pH 7.4, centrifuged as above,and resuspended in 50 mM Tris for immediate use in adenylate cyclaseassay (below). For binding assays, the membrane preparation was thawed,centrifuged (24,000 g×20 min) and resuspended in Tris-isosaline exceptwhere indicated. Aliquots of membrane preparation were added to tubescontaining 50 mM Tris, pH 7.4, 0.9% NaCl, 0.025% ascorbic acid, 0.001%bovine serum albumin, ³H-spiperone and indicated drugs. (+)-Butaclamol(2 μM) was used to define nonspecific binding, which was less than 10%of total binding at concentrations of radioligand near the K_(D) value.Assays were carried out in duplicate, in a volume of 2 ml for saturationanalyses or 1 ml for inhibition analyses. Incubations were initiated byaddition of 10-50 μg of membrane protein, carried out at 37° C. for 50min, and stopped by addition of 10 ml of ice-cold buffer (10 mM Tris, pH7.4, 0.9% NaCl) to each tube. The samples were immediately filteredthrough glass-fibre filters (Schleicher and Schuell No. 30) and washedwith 10 ml of ice-cold buffer. Radioactivity retained on the filter wascounted using a Beckman LS 1701 scintillation counter. In experiments toexamine the effect of GTP on dopamine binding, cells were harvested,centrifuged, resuspended in Tris-Mg⁺⁺ (50 mM Tris, pH 7.4, 4 mM MgCl₂)and incubated for 15 min, 37° C. After centrifugation, the resuspendedprotein was added to assays containing Tris-Mg⁺⁺ with no added GTP orNaCl, or Tris-Mg⁺⁺ with 120 ²Q mM NaCl and 100 μm GTP.

[0198] cAMP and PRL Assay: Cells were plated in 6-well, 35 mm dishes,3-7 days prior to experimentation. Cells were pre-incubated in 2 ml/wellwarm F10+0.1% (−)-ascorbic acid +20 mM Tris (pH 7.2) (FAT) for 5-10 min,followed by addition of 1 ml/well of FAT+100 μm IBMX+experimentalcompounds, and incubated for 30 min. at 37° C. Experimental compoundswere diluted 200- to 1000-fold from stock solutions made immediatelyprior to assay. The final ethanol concentration never exceeded 0.1%, aconcentration without effect on basal or VIP-enhanced cAMP or PRL levelsin GH₄ZR₇ cells. Media were collected, and the cells were lysedimmediately in 1 ml of boiling water. Cell lysates and media werecentrifuged (2000×g, 10 min., 4° C.), and the supernatants collected forassay as cell extracts. Cell extracts and media samples were frozen at−20° C. until assay, if not assayed immediately. cAMP was assayed by aspecific radioimmunoassay as described (22), with antibody used at 1:500dilution. After 16 h incubation at 4° C., 20 μl of 10% BSA and 1 ml of95% ethanol were added consecutively to precipitate the antibody-antigencomplex. Standard curves showed IC₅₀ of 0.5±0.2 pmol using cAMP asstandard. PRL was assayed in the media samples obtained as describedabove, except that IBMX was omitted during the 30 min incubation in FATmedium. PRL levels were determined by specific radioimmunoassay usingStaphyloccus A lysate (Igsorb, The Enzyme Center, Malden, Mass.) toprecipitate antigen-antibody complexes (23). Standard curves gave IC₅₀of 36±6 ng using rPRL standard.

[0199] Adenylate Cyclase Assay: The conversion of α³²P-ATP to ³²P-cAMPwas determined essentially as described by Salomon et al. (24).Membranes (10-50 μg) were added in a volume of 100 μl to an assay of 200μl containing 50 mM Tris, pH 7.4, 5 mM cAMP, 1 mM IBMX, 1 mM MgCl₂, 0.5mM EGTA, 0.25 mM ATP, 30 μm GTP, and about 2×10⁶ cpm of α³²P-ATP, andvarious drugs. Assays carried out in triplicate were initiated bywarming to 25° C. and terminated by cooling to 0° C. Trichloroaceticacid (100 μl of 30% solution) was added to each assay, and ³H-cAMP(30,000 cpm) was added to each tube as an internal standard. The assayvolume was increased to 1 ml by addition of water, and tubes werecentrifuged (2000g×10 min). cAMP in the supernatant was isolated bysequential chromatography on columns containing Dowex AG50W-X4 resin andneutral alumina. The 2-ml eluate from each alumina column was dissolvedin 10 ml of Bio-Safe II (RPI, Mount Prospect, Ill.) for liquidscintillation counting.

[0200] Calculations: Data from cAMP and PRL assays are expressed asmeans±standard error for triplicate determinations. Curve-fittingparameters were obtained by nonlinear regression analysis using theEnzfitter program (Elsevier Biosoft). Average affinity, EC₅₀ and IC₅₀values are geometric means of the indicated number of experiments. Incompetition experiments, K_(I) values were calculated fromexperimentally determined IC₅₀ values by the method of Cheng and Prusoff(25). All experiments were representative of 3-5 independent trials,with the exception of that presented in FIG. 16 (2 trials).

[0201] Results

[0202] Characterization of Stable Transfectants: GH₄C₁ cells werecotransfected with pZEM-D₂-cDNA and pRSV-neo, and colonies resistant tothe antibiotic G418 were isolated and initially characterized byNorthern blot analysis. One clone, GH₄ZR₇, had higher levels of 2.5 kbD₂ mRNA than other clones (FIG. 13A). Wild-type (untransfected) GH₄C₁cells, as well as a GH₄C₁ cell transfectant (GH₄ZD₁₀) expressing the rat5-HT_(1A) receptor gene in the pZEM-3 vector³ showed no hybridization tothe D₂-receptor probe. Pretreatment of GH₄ZR₇ cells with 100 μM ZnSO₄for 36 h induced a marked enhancement of D₂ receptor mRNA, indicatingthat the transcribed mRNA is regulated by the zinc-sensitivemetallothionein promotor (20). The GH₄ZR₇ clone was used for furtheranalysis (below) because of the high levels of dopamine-D₂ receptorexpression in this clone.

[0203] Specific binding of the selective dopamine-D2 receptorantagonist, ³H-spiperone, was assayed in crude membranes prepared fromGH₄ZR₇ cells (FIG. 13B). The GH₄ZR₇ membranes showed a saturablecomponent of ³H-spiperone binding which was displaced by 2 μM(+)-butaclamol, whereas membranes from wild-type GH₄C₁ cells showed nospecific ³H-spiperone binding (data not shown). In 5 experiments, theGH₄ZR₇ cell membranes showed maximal specific ³H-spiperone binding of2046±315 fmol/mg of protein, and a mean K_(D) value of 96±1 pM. Thesevalues demonstrate robust expression of dopamine-D₂ binding sitesreceptors in these cells with affinity for ³H-spiperone comparable tothat obtained in rat striatal membranes, and in Ltk⁻ cells transfectedwith pZEM-D₂-cDNA (17).

[0204] To ascertain whether the expressed dopamine binding siteinteracted with a G protein, inhibition of ³H-spiperone binding bydopamine was assayed in GH₄ZR₇ cell membranes, in the absence orpresence of 100 μM GTP and 120 m!M NaCl (FIG. 13C). Assays were carriedout in the presence of 4 mM MgCl₂ to promote high affinity binding ofdopamine. In the absence of added GTP and NaCl, dopamine inhibited³H-spiperone binding with IC₅₀=49±15 μM, and Hill coefficient of 0.69,suggesting the presence of high and low affinity sites for dopamine.Analyzing the data according to a model assuming the presence of twoclasses of binding sites indicated that 46±15% of the receptors had ahigh affinity (K_(D)=0.5 μM) for dopamine and the remaining receptorshad lower affinity (30 μM) for the agonist. In the presence of GTP andNaCl, the IC₅₀ for dopamine was shifted two-fold to 109±20 μM (K_(I)=17AM) with Hill coefficient closer to unity (0.93). Thus, the presence ofGTP/NaCl converts the dopamine receptors from a heterogeneous populationof high and low affinity receptors to a nearly homogeneous population ofreceptors in a low-affinity agonist state, as observed in striatalmembrane preparations (6, 7). These data suggest that the cloneddopamine binding site interacts with G proteins when expressed in GH₄cells.

[0205] Dopamine Actions on cAMP and PRL Levels: To test directly thefunction of the expressed dopamine-D₂ receptor clone, the actions ofdopamine on cellular cAMP levels were measured. These assays wereconducted in the presence of 100 μM IBMX, to inhibit phosphodiesteraseactivity in these cells (22). Thus, the observed changes in cAMP levelsreflect changes in the rate of synthesis of cAMP rather than changes inits degradation. Dopamine actions on basal cAMP levels were measured, aswell as dopamine inhibition of VIP-enhanced levels of cAMP. GH₄C₁ cellsrespond to VIP with an enhancement of cAMP accumulation (FIG. 14A) asdescribed by others (22, 26). Dopamine had no effect on extracellularcAMP levels in wild-type GH₄C₁ cells, whether VIP was omitted or presentduring the incubation. This result is consistent with the lack ofD₂-dopamine receptor mRNA and binding in GH₄C₁ cells (FIG. 13), andindicates that these cells also lack a detectable D₂-dopamine responsesince dopamine does not elevate cAMP concentrations. In media fromGH₄ZR₇ cells, dopamine inhibited both basal cAMP levels (by 50-70%), andreduced VIP-enhanced cAMP to basal levels. These actions of dopamine toreduce cAMP levels were consistently observed in all experiments anddopamine was equally effective in lowering intracellular cAMP levels(FIG. 14B). As observed previously in GH₄C₁ cells (22), both intra- andextracellular cAMP levels change in parallel, although changes inextracellular cAMP may be more pronounced due to lower recovery ofextracted intracellular cAMP. Dopamine actions on cAMP accumulation wereblocked by (−)-sulpiride, a highly selective dopamine-D₂ antagonist,whereas the inactive stereoisomer, (+)-sulpiride, did not blockdopaminergic inhibition of cAMP accumulation. Stereo-selective blockadeby sulpiride suggested that inhibition of cAMP levels in GH₄ZR₇ bydopamine was mediated by activation of a dopamine-D₂ receptor notpresent in wild-type GH₄C₁ cells.

[0206] The physiological outcome of dopamine action is inhibition ofsecretion, which was assayed by measuring acute (30 min) PRL release inGH₄ZR₇ cells (FIG. 14D). VIP and TRH enhanced PRL secretion 1.5- and3-fold, respectively. VIP is thought to enhance PRL release by acAMP-dependent mechanism (22, 23, 26), while TRH acts by acAMP-independent mechanism linked to calcium mobilization (19, 27).Dopamine did not inhibit basal PRL release, but both VIP- andTRH-induced enhancement of PRL secretion were blocked by dopamine. Thus,dopamine blocked both cAMP-dependent and cAMP-independent secretion inGH₄ZR₇ cells. These actions of dopamine were reversed by (−)-sulpiride,but not by (+)-sulpiride. In GH₄C₁ cells, dopamine had no effect onbasal, VIP-stimulated, or TRH-stimulated secretion of PRL (data notshown).

[0207] To examine whether concentrations required for biologicalresponse correlated with affinity for the dopamine-D₂ receptor,dose-response relations were examined for dopamine actions on cAMPlevels (FIG. 15). Dopamine potently inhibited intra- and extracellularlevels of cAMP with similar EC₅₀ values. Furthermore, dopamine inhibitedboth basal and VIP-enhanced cAMP accumulation with EC₅₀ values of 8±2 nMand 6±1 nM, respectively. These data demonstrate that dopamine inhibitsboth basal and stimulated cAMP accumulation with approximately equalpotency. The high potency of these inhibitory actions of dopaminesupports the assertion that GH₄ZR₇ cells express a functionaldopamine-D₂ receptor.

[0208] The pharmacological specificity of dopaminergic inhibition ofVIP-enhanced levels of cAMP in GH₄ZR₇ was examined further usingspecific receptor antagonists (FIG. 16). The data show that certainreceptor antagonists reverse dopamine-induced inhibition of VIP-enhancedlevels of extracellular cAMP. Maximal cAMP (100%) corresponded to cAMPlevels in the presence of VIP alone. Low concentrations of dopamine-D₂antagonists (spiperone, (+)-butaclamol, (−)-sulpiride) blocked dopamineaction, whereas SCH23390, a specific dopamine-D₁ antagonist, was activeonly at very high concentrations. Inactive stereoisomers ofD₂-antagonists ((−)-butaclamol, (+)-sulpiride (FIG. 14C)) had little orno effect on dopamine action. Antagonists added in the absence ofdopamine did not alter cAMP concentrations. Estimated K_(I) valuesobtained from IC₅₀ values for the antagonists (see description of FIG.16) were similar to values determined from binding competition studiesof the dopamine D₂ receptor (17), showing that inhibition of cAMP levelsby dopamine in GH₄ZR₇ cells is mediated by a receptor which ispharmacologically indistinguishable from the dopamine-D₂ receptor.

[0209] Inhibition of Adenylate Cyclase: To assess directly inhibition ofadenylate cyclase activity by dopamine receptor agonists, the conversionof ³²P-ATP to ³²P-cAMP was measured in membranes prepared from GH₄ZR₇cells (FIG. 17). Dopamine inhibited total forskolin (10 μM)-stimulatedactivity by 45% with an average EC₅₀ value of 0.36 μM (n-5). As observedin pituitary (11) and striatal (28) membranes, bromocryptine behaved aspartial agonist, maximally inhibiting enzyme activity by 23% (EC₅₀=6nM). Inhibition of adenylate cyclase activity by selective D₂-agonistswas stereo-selective. For example, quinpirole inhibitedforskolin-stimulated cyclase activity by 41% (EC₅₀=0.32 nM), whereasLY181990, the inactive (+)-enantiomer of quinpirole, cause no consistentreduction in enzyme activity. Similarly, (+)-3-PPP (EC₅₀=0.86 nM) was asefficacious as dopamine, whereas the enantiomer (−)-3-PPP did notconsistently educe adenylate cyclase activity. VIP also stimulatedadenylate cyclase activity in GH₄ZR₇ cell membranes, as reported forwild-type GH₄C₁ cell membranes (29). Total activity stimulated by 200 nMVIP was 22±7 pmol/mg protein/min (n=3), and VIP-enhanced activity wasinhibited 41% by dopamine (100 μM), compared to 50-55% inhibition in ratanterior pituitary membranes (11). No effect of dopamine on basaladenylate cyclase activity was observed in these preparations.Nevertheless, inhibition by dopamine of forskolin- or VIP-stimulatedadenylate cyclase activity provides a likely mechanism for inhibition ofcAMP accumulation by dopamine in GH₄ZR₇ cells.

[0210] Pertussis Toxin Sensitivity: Sensitivity to pertussis toxin is ahallmark of receptors, such as the dopamine-D₂ receptor (6-12, 15),which couple to inhibitor G proteins (e.g., G_(i) or G_(o)) to induceresponses. Pretreatment of GH₄ZR₇ cells with pertussis toxin for 16 h(FIG. 12) uncoupled dopamine-mediated inhibition of forskolin-stimulatedmembrane adenylate cyclase activity, and abolished inhibition of basaland VIP-stimulated cAMP accumulation by dopamine. The concentration ofpertussis toxin and incubation time used produce maximal blockage ofsomatostatin responses in wild-type cells (30), and the dopamineresponses were almost completely inhibited under these conditions. Bycontrast, basal and VIP-stimulated cAMP accumulation, as well as basaland forskolin-stimulated cyclase activity, were not significantlyaltered by pertussis toxin pretreatment. These data support theassertion that the expressed cDNA clone codes for a dopamine-D₂ bindingsite which is functionally coupled to inhibitory G proteins present inGH₄ cells, and thus represents a bona fide receptor.

[0211] Discussion

[0212] The cDNA clone coding for a dopamine-D₂ binding site (17) wasexpressed in GH₄C₁ cells to determine whether the lone expresses afunctional D₂ receptor, which is coupled by pertussis toxin-sensitiveinhibitory G proteins to inhibition of adenylate cyclase, cAMPaccumulation, and inhibition of PRL secretion (6-12, 15). Dopamine-D2receptors were expressed specifically from a D₂-cDNA construct under theregulation of the mouse metallothionein promotor (20), as evidenced bythe presence of D₂ receptor mRNA in the GH₄ZR₇ transfectant. Dopamine-D₂receptor mRNA levels were undetectable in untransfected GH₄C₁ cells, orin cells transfected with the rat 5-HT_(1A) receptor subtype (FIG. 13A).The mRNA species found in GH₄ZR₇ cells was approximately the samemolecular weight as D₂ receptor mRNA found in rat brain (17). Levels ofdopamine-D₂ mRNA increased by addition of 100 μM Zn⁺⁺ in GH₄ZR₇ cells,indicating that expression of the mRNA is controlled by theZn⁺⁺-sensitive mouse metallothionein promotor, and does not representtranscription of the endogenous gene. Specific binding of ³H-spiperonebinding was present only in GH₄ZR₇ cells, and was increased by 100 μMZn⁺⁺, and thus correlated with the expression of dopamine-D₂ receptormRNA in these cells. The D₂-receptor mRNA was transcribed to yieldrobust expression of high affinity dopamine-D₂ binding sites in GH₄ZR₇cells.

[0213] The presence of dopamine-D₂ binding in the GH₄ZR₇ transfectantcorrelated with potent and powerful inhibition of cAMP accumulation andPRL release, as well as inhibition of forskolin-stimulated adenylatecyclase activity, actions of dopamine not observed in untransfectedGH₄C₁ cells. These inhibitory actions of dopamine match exactly theknown physiological actions of dopamine in pituitary lactotrophs (1, 2).In particular, dopamine controls PRL secretion and cAMP accumulation inlactotrophs such that stimulation of these processes does not occurunless dopamine concentrations decrease to low levels (1, 3-5).Similarly, in the present of maximal concentrations of dopamine, VIPdoes not enhance cAMP levels or PRL secretion in GH₄ZR₇ cells (FIG. 14).The potency of dopamine inhibition of basal and VIP-enhanced cAMPaccumulation in GH₄ZR₇ cells (FIG. 15) was in the range of concentrationexpected for lactotrophs, given that dopamine concentrations inhypophyseal portal blood vary from 7 nM in female rates duringproestrous, to 20 nM during estrous, and are 3 nM in male rats (31).Detailed analysis of the pharmacology of dopamine-induced inhibition ofadenylate cyclase and cAMP accumulation using specific agonists andantagonists are fully consistent with the conclusion that dopaminergicactions in GH₄ZR₇ cells are mediated by a receptor indistinguishablefrom the dopamine-D₂ receptor.

[0214] The discrepancy between the measured affinity of dopamine (FIG.13C), and the potency of dopamine to inhibit cAMP accumulation (FIG. 13)raises the possibility that GH₄ZR₇ cells have “spare” receptors, i.e., asufficient excess of binding sites to shift the EC₅₀ for biologicalaction to values lower than the K_(D) value. An alternative explanationis that the receptor in membrane preparations has a lower affinity foragonists (but unchanged affinity for antagonists since measured IC₅₀values correlated with K₁ values) than in intact cells. Since cytosolicor membrane-associated components present in intact cells are notentirely replaced in membrane binding and adenylate cyclase assayconditions, it is possible that components which allow for optimalfunction of the dopamine receptor in membrane preparations are lacking.This assertion is supported by the EC₅₀ value for inhibition ofparticulate adenylate cyclase by dopamine (360 nM), which is close toK_(I) values obtained for dopamine from binding competition experiments(500 nM), but 100-fold higher EC₅₀ values (6-8 nM) obtained forinhibition of cAMP levels by dopamine in intact cells. This differencein conditions may explain the observed differences between assays inintact versus particulate preparations. However, affinities ofantagonists correlated well with estimated K_(I) values obtained fromcAMP accumulation experiments (FIG. 16), indicating that antagonistbinding is similar in membranes and whole cells.

[0215] Further evidence of coupling of the expressed dopamine-D₂ bindingsite to G proteins is the shift of dopamine binding affinity to loweraffinity in the presence of GTP. Such GTP-induced shifts in affinityhave been reported for dopamine binding in membranes from rat brain (6,7), and are due to interaction of the receptor with G proteins (8, 9).In the presence of GTP, the G protein dissociates from the receptorleaving the receptor in a low affinity agonist state (6, 7). In the caseof dopamine, the difference between the affinities of the two states issmall, hence the GTP-induced shift to the low-affinity state is small(two-fold) and requires the presence of Na⁺ ion to maximize dissociationof the G protein (28). The observation of a GTP-induced shift indopamine affinity suggests that the expressed dopamine receptor isassociated with one or more G proteins in GH₄ZR₇ cell membranes.

[0216] Although the inhibitory actions of dopamine indicate that thereceptor couples to inhibitory G proteins, this was tested more directlyby pretreating GH₄ZR₇ cells with pertussis toxin to inactivateinhibitory G proteins (30). Pertussis toxin pretreatment completelyblocked dopamine actions, without altering basal or VIP-stimulated cAMPaccumulation. Thus, pertussis toxin prevented the dopamine-enhancedtransduction of biological responses, presumably by uncoupling thedopamine-D₂ receptor from inhibitory G proteins. Unlike other systems(32), but as seen in wild-type GH₄C₁ cells (30), enhancement of cAMPlevels by stimulators (e.g., VIP) was not augmented in pertussistoxin-treated GH₄ZR₇ cells, nor were basal cAMP levels altered bypertussis toxin. This suggests that inhibitory G proteins present inGH₄ZR₇ cells do not inhibit cAMP generation tonically.

[0217] The presence of somatostatin receptors on GH₄C₁ cells (33) allowsfor direct comparison of the inhibitory actions of somatostatin anddopamine. Like dopamine, somatostatin does not inhibit basal adenylatecyclase activity (29) or basal PRL secretion (30). Somatostatin-inducedinhibition of VIP-stimulated cyclase activity (29), VIP-enhanced cAMPaccumulation (22), and PRL secretion (23, 30) in GH₄C₁ cells wereall-about half the maximal inhibition induced by dopamine in GH₄ZR₇cells. Indeed, in GH₄ZR₇ cells, somatostatin was half as effective asdopamine at inhibiting VIP-enhanced cAMP accumulation (data not shown).The larger effects of dopamine were not due to the presence of anexcessive number of D₂ receptors since the receptor number underconditions of cAMP accumulation and PRL secretion experiments was lessthan 10,000 sites/cell, smaller than somatostatin receptor number(13,000 sites/cell) in GH₄C₁ cells (33). It is apparent that thedopamine receptors expressed in these cells are more effective attransducing inhibitory actions than somatostatin receptors, suggesting amore effective coupling of the dopamine receptor to the G proteinspresent in GH cells.

[0218] A second reason for expressing the dopamine-D₂ receptor cDNA inGH₄C, cells was to establish a new model system in which to studydopamine actions and D₂ receptor mechanisms. Current studies on dopamineaction in rat lactotrophs, the most accessible cell system expressingdopamine-D₂ receptors until recently (16), have come to divergentconclusions on mechanisms of dopamine inhibition. In neurons, dopaminehyperpolarizes membrane potential (34) by opening potassium channels(35), actions mediated by coupling of the D2 receptor to inhibitory Gproteins (15). Membrane hyperpolarization induced by dopamine inlactotrophs (36) would close calcium channels, decreasing basal calciuminflux and explaining observed decreases in basal [Ca⁺⁺]_(i) induced bydopamine (13-15, 37, 38). However, dopamine has been observed toincrease [Ca⁺⁺]_(i) in certain pituitary cells (37). By examiningdopamine-induced changes in [Ca⁺⁺]_(i) in GH₄ZR₇ cells it will bepossible to specifically associate the D₂ receptor with changes in[Ca⁺⁺]_(i), and to ascertain the role of [Ca⁺⁺]_(i) in mediatingdopamine inhibition of hormone secretion. Another unresolved issue isthe mechanism by which dopamine inhibits enhancement of secretion and bycalcium-mobilizing hormones such as TRH. While some report inhibition bydopamine of TRH-induced enhancement of phosphatidyl inositol turnover(39, 40) and [Ca⁺⁺]_(i) (13, 14), others find no change (38, 41). Theobserved inhibition by dopamine of TRH-induced PRL release in GH₄ZR₇cells (FIG. 14C) suggests that dopamine may be coupled by G proteins toprocesses (e.g., opening of potassium channels) which alter TRH-inducedcalcium-mobilization or phosphatidyl inositol turnover. GH₄ZR₇ cellswill provide a homogeneous, abundant, and highly-responsive preparationin which to study these and other questions regarding mechanisms of thedopamine-D₂ receptor.

[0219] The data presented in this report indicate that the expressedclone (17) possesses the pharmacology of the dopamine-D2 receptor in allactions investigated including inhibition of PRL secretion, cAMPgeneration, and adenylate cyclase activity. The D₂ clone meets fivebasic criteria for classification as a functional G protein-coupledreceptor: 1) the cDNA clone for the dopamine-D₂ binding site possessesthe archetypical structure of G protein-coupled receptors; 2) the cloneexpresses a protein with saturable and specific binding properties; 3)agonist binding affinity to the expressed binding site is decreased inthe presence of GTP; 4) the expressed receptor is coupled to functions(e.g., inhibition of adenylate cyclase) known to be regulated by Gproteins; 5) agents (e.g., pertussis toxin) which uncouple G proteinfunction uncouple activation of the expressed receptor from generationof the appropriate response. In conclusion, the interaction of thecloned dopamine-D₂ receptor with G proteins is productive, leading toactivation of α_(i) or α_(o) subunits and consequent inhibition of cAMPand PRL levels by both cAMP-dependent and cAMP-independent mechanisms.

REFERENCES FOR EXAMPLE 2

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[0223] 4. Lopez, F. J., Dominguez, J. R., Sanchez-Criado, J. E., andNegro-Vilar, A. (1988), Endocrinology 124, 527-535.

[0224] 5. Ibid, pp. 536-542.

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[0228] 9. Ohara, K., Haga, K., Berstein, G., Haga, T., Ichiyama, A., andOhara, K. (1988), Mol. Pharmacol. 33, 290-296.

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[0230] 11. Onali, P., Schwartz, J. P., and Costa, E. (1981), P.N.A.S.78, 6531-6534.

[0231] 12. Cronin, M. J., Myer, G. A., MacLeod, R. M., and Hewlett, E.(1983), Am. J. Physiol. 244, E499-E504.

[0232] 13. Schofield, J. G. (1983), F.E.B.S. Lett. 159, 79-82.

[0233] 14. Margaroli, A., Vallar, L., Elahi, F. R., Pozzan, T., Spada,A., and Meldolesi, J. (1987), J. Biol. Chem. 261, 13920-13927.

[0234] 15. Vallar, L. and Meldolesi, J. (1989), TIPS 10, 74-77.

[0235] 16. Judd, A. M., Login, I. S., Kovacs, K., Ross, P. C., Spangelo,B. L., Jarvis, W. D., and MacLeod, R. M. (1988), Endocrinology 123,2341-2350.

[0236] 17. Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P.,Salon, J., Christie, M., Machida, C., Neva, K. A., and Civelli, O.(1988), Nature 336, 783-787.

[0237] 18. Tashjian, A. H., Jr. (1979), Meth. Enzymol. 58, 526-535.

[0238] 19. Ozawa, S., and Sand, 0. (1986), Physiol. Rev. 66, 887-952.

[0239] 20. Uhler, M., and McKnight, G. S. (1987), J. Biol. Chem. 262,15202-15207.

[0240] 21. Chirgwin, J. M., Prybyla, A. E., MacDonald, R. J., andRutter, W. J. (1979).

[0241] 22. Dorflinger, L. J., and Schonbrunn, A. (1983), Endocrinology113, 1541-1550.

[0242] 23. Dorflinger, L. J., and Schonbrunn, A. (1983), Endocrinology113, 1551-1558.

[0243] 24. Salomon, Y., Londos, C., and Rodbell, M. (1974), Analyt.Biochem. 58, 541-548.

[0244] 25. Cheng, Y. -C., and Prusoff, W. H. (1973), Biochem. Pharmacol.22, 3099-3108.

[0245] 26. Gourdji, D., Bataille, D., Vauclin, N., Grouselle, D.,Rosselin, G., and Tixier-Vidal, A. (1979), FEBS Lett. 104, 165-168.

[0246] 27. Albert, P. R., and Tashjian, A. H., Jr. (1984), J. Biol.Chem. 259, 15350-15363.

[0247] 28. Onali, P., Olianas, M. C., and Gessa, G. L. (1985), Mol.Pharmacol. 28, 138-145.

[0248] 29. Koch, B. D., and Schonbrunn, A. (1984), Endocrinology 114,1784-1790.

[0249] 30. Koch, B. D., Dorflinger, L. D., and Schonbrunn, A. (1985), J.Biol. Chem. 260, 13138-13145.

[0250] 31. Ben-Jonathan, N., Oliver, C., Weiner, H.J., Mical, R. S., andPorter, J. C. (1977), Endocrinology 100, 452-458.

[0251] 32. Kurose, H., Katada, T., Amano, T., and Ui, M. (1983), J.Biol. Chem. 258, 4870-4875.

[0252] 33., Schonbrunn, A., and Tashjian, A. H., Jr. (1978), J. Biol.Chem. 253, 6473-6483.

[0253] 34. Bunney, B. S., Aghajanian, G. K., and Roth, R. H. (1973),Nature (New Biol.) 245, 123-125.

[0254] 35. Lacey, M. G., Mercuri, N. B., and North, R. A. (1987), J.Physiol. 392, 397-416.

[0255] 36. Taraskevich, P. S., and Douglas, W. W. (1978), Nature 276,832-834.

[0256] 37. Winiger, B. P., Wuarin, F., Zahan, G. R., Wollheim, C. B.,and Schlegel, W. (1987), Endocrinology 121, 2222-2228.

[0257] 38. Law, G. J., Pachter, J. A., and Dannies, P. (1988), Mol.Endocrinol. 2, 966-972.

[0258] 39. Journot, L., Homburger, V., Pantaloni, C., Priam, M.,Bockaert, J., and Enjalbert, A. (1987), J. Biol. Chem. 262, 15106-15110.

[0259] 40. Vallar, L., Vicentini, L. M., and Meldolesi, J. (1988), J.Biol. Chem. 263, 10127-10134.

[0260] 41. Canonico, P. L., Jarvis, W. D., Judd, A. M., and MacLeod, R.M. (1986), J. Endocrinol. 110, 389-393.

FOOTNOTES

[0261]¹ The abbreviations used are: PRL, prolactin; GH, growth hormone;[Ca⁺⁺]_(i), cytosolic free calcium concentration; VIP, vasoactiveintestinal peptide; TRH, thyrotropin-releasing hormone; IBMX,3-isobutyl-1-methyl xanthine; K_(D), equilibrium dissociation constant;EC₅₀ (IC₅₀), concentration required to elicit a half-maximal effect(inhibition).

[0262]² These results have been presented in part at the 71^(st) AnnualEndocrine Meetings, Seattle, Wash., Abstract #1278.

[0263]³ Albert, P. R., et al., manuscript in preparation.

Example 3

[0264] Abstract

[0265] A clone encoding a human dopamine D₂ receptor was isolated from apituitary cDNA library and sequenced. The deduced protein sequence is96% identical with that of the cloned rat receptor [Bunzow et al. (1988)Nature 336, 783-787] with one major difference: The human receptorcontains an additional 29 amino acids in its putative third cytoplasmicloop. Southern blotting demonstrated the presence of only one humandopamine D₂ receptor gene. Two overlapping phage containing the genewere isolated and characterized. DNA sequence analysis of these clonesshowed that the coding sequence is interrupted by six introns and thatthe additional amino acids present in the human pituitary receptor areencoded by a single exon of 87-basepairs. The involvement of thissequence in alternative splicing and its biological significance arediscussed.

[0266] Introduction

[0267] Dopamine neurons in the vertebrate central nervous system areinvolved in the initiation and execution of movement, the maintenance ofemotional stability, and the regulation of pituitary function. Severalhuman neurological diseases, including Parkinson's Disease (1) andschizophrenia (2), are thought to be manifestations of imbalancesbetween dopamine receptors and dopamine. The receptors which mediatedopamine's effects have been divided into D₁ and D₂ subtypes, which aredistinguished by their G-protein coupling (3, 4), ligand specificities,anatomical distribution and physiological effects (5). The dopamine D₂receptors have been of particular clinical interest due to theirregulation of prolactin secretion (6) and their affinity forantipsychotic drugs (7, 8).

[0268] The dopamine D₂ receptors belong to the family of G-proteincoupled receptors. The sequence similarity shared by members of thisfamily enabled us to clone a rat brain dopamine D₂ receptor cDNA (9). Wehave used that clone to isolate the human pituitary dopamine D₂ receptorcDNA described here. We have found that the deduced amino acid sequencesof these two receptors are very similar, with one notable difference.The human receptor contains an additional 29 amino acids in its putativethird cytoplasmic loop which are encoded by one of the gene's exons.This genomic organization suggests that the existence of two dopamine D₂receptor mRNAs is the result of an alternative splicing event.

[0269] Materials and Methods

[0270] Cloning of the Human Pituitary cDNA

[0271] Human pituitary tissue was a generous gift from Drs. N. Seidahand M. Chretien, Clinical Research Institute of Montreal, Canada.Poly(A)⁺ mRNA and cDNA were prepared as previously described (9). ThecDNA was size-selected (1-6 kb) on agarose gels, isolated usingGeneclean (Bio 101), ligated to EcoRI adaptors, cloned into λGT10 arms(Stratagene), and packaged (Gigapak Gold). Recombinants (1.5×10⁶) werescreened on replica nylon filters (DuPont Plaque/Colony Hybridizationfilters) with [³²P]-labelled hybridization probes. Prehybridization andhybridization were performed in 50% formamide, 1% SDS, 2×SSC (1×SSC=0.15M NaCl/0.015 M sodium citrate, pH 7) at 37° C. The complete sequence ofboth strands of DNA were determined in M13 mp19 using Sequenase (U.S.Biochemical) primed with synthetic oligonucleotides.

[0272] Expression and Pharmacology

[0273] The 2.5-kb human pituitary cDNA (hPitD₂) was cloned into pZem3 (agift from Dr. E. Mulvihill, Zymogenetics) and co-transfected with thepRSVneo gene into mouse Ltk⁻ cells by CaPO₄ precipitation (10). A stabletransfectant (L-hPitD₂Zem) was selected and maintained in 750 μg/ml ofG418 (Geneticin sulphate, Gibco). Twenty hours prior to the harvestingof membranes, these cells were incubated with 70 μM zinc sulphate.Membranes were prepared from L-HPitD₂Zem, from the Ltk⁻ cell lineexpressing the cloned rat dopamine D₂ receptor (L-RGB2Zem-1) and fromfreshly dissected rat striata (Taconic Farm, Germantown, N.Y.), aspreviously described (11, 12). For the binding assays, membrane proteinwas used at 10-15 μg from L-hPitD₂Zem, 50-75 μg from L-RGB2Zem-l, and220-250 μg from rat striatum. The binding assays were incubated at 37°C. for 60 minutes in 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂,1 mM MgCl₂, pH 7.4, and then stopped by rapid filtration over glassfiber filters (Schleicher and Schuell, No. 32) which had been presoakedin 0.5% polyethyleneimine. The filters were washed twice in ice cold 50mM Tris-HCl, pH 7.4. Saturation curves were generated using increasingconcentrations of the highly D₂-specific antagonist [³H]-domperidone(13). Antagonist drugs were evaluated for their ability to inhibitspecifically bound [³H]-domperidone (1 nM). The B_(max), K_(d), and IC₅₀values were determined as previously described (14).

[0274] Southern Blotting

[0275] Human genomic DNA prepared from a normal male donor was a giftfrom M. Litt. Three micrograms of DNA were digested with restrictionenzymes and the fragments were electrophoresed in 0.7% agarose andblotted onto nitrocellulose filters (Schleicher and Schuell).Prehybridization and hybridization were performed at 37° C. in 50%formamide as previously described (15).

[0276] Genomic Sequencing

[0277] Genomic bacteriophage lambda libraries, prepared from normalhuman male DNA, were purchased from Stratagene and ClontechLaboratories, Inc. and screened with portions of the cloned rat dopamineD₂ receptor cDNA. DNA sequence was determined by a genomic sequencingapproach (16, 17). Briefly, for each restriction enzyme used, clonedgenomic phage DNA (50 μg) was digested, subjected to chemical cleavage,and the resulting fragments resolved in a denaturing polyacrylamide gel.The DNA was then transferred from the gel and immobilized onto nylonfilters (Plasco Genetran). Using [³²P] end-labelled synthetic oligomers,ladders of sequence were visualized within exons and read intoneighboring introns. The filters were then either reprobed with adifferent oligomer, or a new filter was made in order to read thecomplementary sequence back across the exon. Both strands of the codingregion were sequenced.

[0278] Results

[0279] Cloning and Sequence Analysis of the Human Pituitary cDNA

[0280] Using the rat brain D₂ receptor cDNA as probe, three partialcDNAs were isolated from a human pituitary library and sequenced. Twooligonucleotide probes based on these sequences were used to isolate afourth cDNA, hPitD₂, which encoded a full-length receptor protein (FIG.18). The human pituitary receptor contains seven putative transmembranedomains and lacks a signal sequence. Overall, the human and ratnucleotide sequences are 90% similar and show 96% identity at the aminoacid level. Several consensus sequences for N-linked glycosylation,protein kinase A phosphorylation and palmitoylation (18) are conservedbetween the human and rat receptors. There are also 18 amino aciddifferences (including one deletion) between these proteins, and,strikingly, the human pituitary receptor contains an additional 29 aminoacids in its putative third cytoplasmic loop.

[0281] Expression and Pharmacological Evaluation of hPit D₂ cDNA

[0282] In order to evaluate the pharmacological characteristics of thehuman pituitary receptor, its cDNA was subcloned into pZem3 andexpressed in mouse Ltk⁻ cells (L-hPitD₂Zem). Membranes prepared fromthese cells showed specific binding of [³H]-domperidone, a D₂-selectiveantagonist (12, 13), with a B_(max) of 4.05+/−0.3 pmol per mg (n=2)protein and a K_(d) of 0.74+/−0.11 nM (n=2). This K_(d) value is inexcellent agreement with the published value of 0.74 nM in mouse brainmembranes (13). A Scatchard plot of the data was linear. There was nodetectable [³H]-domperidone binding in membranes prepared from cellstransfected with pZem3 alone (data not shown). [³H]-domperidone bindingto L-hPitD₂Zem membranes was inhibited by a number of dopamineD₂-specific drugs (FIG. 19). Their rank order of potency was: spiperone,(+)-butaclamol, haloperidol, and sulpiride. The serotonin-selectiveantagonist mianserin and the D₁-selective antagonist SCH-23390 inhibiteddomperidone binding only at very high concentrations, as did theinactive isomer (−)-butaclamol. These values are essentially identicalto those obtained with membranes from Ltk⁻ cells transfected with thecloned rat dopamine D₂ receptor cDNA (L-RGB2Zem-1) and from rat striatum(FIG. 22).

[0283] Human Dopamine D₂ Receptor Gene

[0284] Using portions of the rat cDNA as probe, a clone was isolatedfrom a human genomic library. This genomic clone, λHD2G1, contained a1.6-kb BamHI fragment which encoded the last 64 amino acids of the humanD₂ receptor and 1.2-kb of 3′ non-coding sequence. The 1.6-kb fragmentwas used to probe a Southern blot of human genomic DNA digested withthree restriction enzymes. Each enzyme generated a single fragment thathybridized to the probe (FIG. 20), indicating that there is probablyonly one human dopamine D₂ receptor gene.

[0285] In order to isolate a genomic clone that encoded the N-terminusof the human receptor protein, a 118-bp restriction fragment from thecloned rat dopamine D₂ receptor cDNA (corresponding to amino acidresidues 1-39) was used to screen a second genomic library. λHD2G2 wasisolated and found to overlap with λHD2G1 by 400 nucleotides (FIG. 21a).Together, these phage span 34-kb of the human dopamine D₂ receptor genelocus, DRD2 (19), and contain the sequence found in the hPitD₂ cDNA plussequences that extend 15-kb downstream of the polyadenylation signal and3.7-kb upstream of the translation initiation site. To characterize theintron/exon structure of the gene, a genomic sequencing approachemploying oligonucleotide probes and chemical cleavage was used (FIG.21b). Since the divergence of nucleotide sequences between human and ratmembers of this receptor family is approximately 10% (unpublishedobservations), we were able to initiate the genomic sequencing relyingon hybridization probes and restriction sites that are present in thecloned rat dopamine D₂ receptor cDNA. our results demonstrate that thecoding portion of the human dopamine D2 receptor gene is divided intoseven exons (FIG. 21b). Interestingly, we found that exon five is 87-bplong and encodes the entire 29 amino acid sequence present in the clonedhuman pituitary receptor (FIGS. 18 and 21c). Analysis of the six intronsrevealed that each contains acceptor and donor sequences that conform tothe GT/AG rule (20), as summarized in FIG. 23. The approximate sizes ofthe introns were based on the results of Southern blotting experiments(data not shown). When compared, the genomic and cDNA sequences werefound to differ by only two silent transitions, one at 939 (T to C inthe gene) and the other at 957 (C to T in the gene).

[0286] Discussion

[0287] Several alternative hypotheses might account for the extrasequence present in our human pituitary D₂ receptor cDNA clone. Onepossibility is that the human gene contains the extra 87 bases and thatthe rat gene does not. Another is that both human and rat have twodistinct genes which code for two different dopamine D₂ receptors.Finally, alternative splicing of a single transcript could result in onemRNA and the other without the 87 bases. In support of the latterhypothesis, we have shown that there is probably only one human dopamineD₂ receptor gene, DRD2, and that the 87-bp sequence is contained on adistinct exon of that gene. Furthermore, we have cloned a rat brain cDNAthat contains the 87-bp sequence (unpublished results). This sequence ishighly similar to that of the human cDNA and established that dopamineD₂ receptors containing the 29 residues are not unique to the humanpituitary.

[0288] The human dopamine D₂ receptor expressed in L-hpitD₂Zem cells hasessentially the same drug binding profile as do rat striatum andL-RGB2Zem-1 membranes. Therefore, since the 29 amino acids do not affectbinding, they may be involved in other levels of receptor function. Forexample, the third cytoplasmic loop of the β₂-adrenergic receptor hasbeen shown to be required for appropriate G-protein coupling (21).Therefore, one possibility is that this sequence may influence whetherdopamine D₂ receptor stimulation inhibits adenylyl cyclase, activatespotassium channel conductance or inhibits calcium mobilization (22).Another possibility is that the 29 amino acids differentiatepost-synaptic dopamine D₂ receptors from presynaptic autoreceptors (23).Since a computer search (VAX/Intelligenetics) failed to identify anothersequence of significant homology, we consider this sequence to be uniqueto the D₂ receptor.

[0289] Based on the comparison of genomic and cDNA sequences, the humandopamine D₂ receptor gene is divided into at least seven exons. It ispossible that one or more additional exons remain to be identified atthe 5′ end of the gene, as was shown to be the case with muscarinicreceptor genes (24).

[0290] The interruption of coding sequence by introns distinguishes thehuman dopamine D₂ receptor gene from most other members of the G-proteincoupled receptor gene family with the exception of the opsin genes (25).One significant observation is that the placement of two introns (Nos. 3and 5) in this human gene corresponds almost precisely to intronpositions conserved in bovine and Drosophila opsin genes (26, 27). Thesimplest interpretation of this finding is that their common ancestor, agene rougly one billion years old (28), contained these introns. Ourcharacterization of the gene structure also provides evidence that theexons encode recognizable elements of protein structure (29). Thatintrons are found following transmembrane segments II, III, and IV (SeeFIGS. 18 and 21) argues that the repeated structural motifscharacteristic of these receptors may have evolved by internalduplication. Furthermore, the presence of several introns within thethird cytoplasmic loop provides an explanation of the substantialvariation in length observed across the family (30).

[0291] The possibility of alternatively spliced dopamine D₂ receptormRNAs giving rise to structurally distinct forms is exciting. Theexpression of one form of the dopamine D₂ receptor mRNA or anotherrepresents a level of control which may have implications with respectto human disease.

Acknowledgments

[0292] We would like to thank Howard Goodman for discussion and reviewof the manuscript, Dee Yarozeski for manuscript preparation, and VickyRobertson, Nancy Kurkinen, and June Shiigi for the illustrations. D.K.G.holds a fellowship from NIH. This work was supported by NIH Grant Nos.Dk37231 and MH45614 and a grant from Cambridge NeuroScience Research,Inc., Cambridge, Mass., to O.C.

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What is claimed is:
 1. An antibody capable of specifically binding to amammalian D2 dopamine receptor having an amino acid sequence identifiedas the amino acid sequence of FIGS. 7A-C, FIGS. 18A-H or FIGS. 18A-Hwherein amino acids 242-270 are deleted therefrom.
 2. An antibodyaccording to claim 1 that is a monoclonal antibody.
 3. Anantigen-binding fragment of an antibody according to claim 1, whereinsaid fragment can be produced by chemical or enzymatic cleavage of saidantibody.
 4. An antigen-binding fragment according to claim 3, whereinthe fragment is an Fab fragment, an F(ab)′ fragment, an F(ab)₂ fragmentor an Fv fragment.
 5. An antibody according to claim 1 wherein themammalian D2 dopamine receptor is a human D2 dopamine receptor.
 6. Anantibody according to claim 5 wherein the mammalian D2 dopamine receptorhas an amino acid sequence identified as the amino acid sequence ofFIGS. 18A-H or FIGS. 18A-H wherein amino acids 242-270 are deletedtherefrom.
 7. An antibody according to claim 1 wherein the mammalian D2dopamine receptor is a rat D2 dopamine receptor.
 8. An antibodyaccording to claim 5 wherein the mammalian D2 dopamine receptor has anamino acid sequence identified as the amino acid sequence of FIGS. 7A-C.9. An antibody according to claim 1 wherein the antibody isdetectably-labeled.
 10. An antibody according to claim 1 where theantibody has immunological binding specificity for an epitope comprisingamino acids 2-13, 182-192, 264-277, 289-298 or 404-414 in FIG. 1.